2-phenoxy pyrimidinone analogues

ABSTRACT

2-Phenoxy pyrimidinone analogues are provided, of the Formula: 
                         
wherein variables are as described herein. Such compounds are ligands that may be used to modulate specific receptor activity in vivo or in vitro, and are particularly useful in the treatment of conditions associated with pathological receptor activation in humans, domesticated companion animals and livestock animals. Pharmaceutical compositions and methods for using such compounds to treat such disorders are provided, as are methods for using such ligands for receptor localization studies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/895,286, filed Aug. 22, 2007, now U.S. Pat. No. 8,003,656, issuedAug. 23, 2011, which claims priority to U.S. Provisional Application60/823,258, filed Aug. 23, 2006. The contents of each of the foregoingapplications are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to 2-phenoxy pyrimidinone analoguesthat have useful pharmacological properties. The invention furtherrelates to the use of such compounds for treating conditions related tocapsaicin receptor activation, for identifying other agents that bind tocapsaicin receptor, and as probes for the detection and localization ofcapsaicin receptors.

BACKGROUND OF THE INVENTION

Pain perception, or nociception, is mediated by the peripheral terminalsof a group of specialized sensory neurons, termed “nociceptors.” A widevariety of physical and chemical stimuli induce activation of suchneurons in mammals, leading to recognition of a potentially harmfulstimulus. Inappropriate or excessive activation of nociceptors, however,can result in debilitating acute or chronic pain.

Neuropathic pain involves pain signal transmission in the absence ofstimulus, and typically results from damage to the nervous system. Inmost instances, such pain is thought to occur because of sensitizationin the peripheral and central nervous systems following initial damageto the peripheral system (e.g., via direct injury or systemic disease).Neuropathic pain is typically burning, shooting and unrelenting in itsintensity and can sometimes be more debilitating that the initial injuryor disease process that induced it.

Existing treatments for neuropathic pain are largely ineffective.Opiates, such as morphine, are potent analgesics, but their usefulnessis limited because of adverse side effects, such as physicaladdictiveness and withdrawal properties, as well as respiratorydepression, mood changes, and decreased intestinal motility withconcomitant constipation, nausea, vomiting, and alterations in theendocrine and autonomic nervous systems. In addition, neuropathic painis frequently non-responsive or only partially responsive toconventional opioid analgesic regimens. Treatments employing theN-methyl-D-aspartate antagonist ketamine or the alpha(2)-adrenergicagonist clonidine can reduce acute or chronic pain, and permit areduction in opioid consumption, but these agents are often poorlytolerated due to side effects.

Topical treatment with capsaicin has been used to treat chronic andacute pain, including neuropathic pain. Capsaicin is a pungent substancederived from the plants of the Solanaceae family (which includes hotchili peppers) and appears to act selectively on the small diameterafferent nerve fibers (A-delta and C fibers) that are believed tomediate pain. The response to capsaicin is characterized by persistentactivation of nociceptors in peripheral tissues, followed by eventualdesensitization of peripheral nociceptors to one or more stimuli. Fromstudies in animals, capsaicin appears to trigger C fiber membranedepolarization by opening cation selective channels for calcium andsodium.

Similar responses are also evoked by structural analogues of capsaicinthat share a common vanilloid moiety. One such analogue isresiniferatoxin (RTX), a natural product of Euphorbia plants. The termvanilloid receptor (VR) was coined to describe the neuronal membranerecognition site for capsaicin and such related irritant compounds. Thecapsaicin response is competitively inhibited (and thereby antagonized)by another capsaicin analog, capsazepine, and is also inhibited by thenon-selective cation channel blocker ruthenium red, which binds to VRwith no more than moderate affinity (typically with a K_(i) value of nolower than 140 μM).

Rat and human vanilloid receptors have been cloned from dorsal rootganglion cells. The first type of vanilloid receptor to be identified isknown as vanilloid receptor type 1 (VR1), and the terms “VR1” and“capsaicin receptor” are used interchangeably herein to refer to ratand/or human receptors of this type, as well as mammalian homologues.The role of VR1 in pain sensation has been confirmed using mice lackingthis receptor, which exhibit no vanilloid-evoked pain behavior andimpaired responses to heat and inflammation. VR1 is a nonselectivecation channel with a threshold for opening that is lowered in responseto elevated temperatures, low pH, and capsaicin receptor agonists.Opening of the capsaicin receptor channel is generally followed by therelease of inflammatory peptides from neurons expressing the receptorand other nearby neurons, increasing the pain response. After initialactivation by capsaicin, the capsaicin receptor undergoes a rapiddesensitization via phosphorylation by cAMP-dependent protein kinase.

Because of their ability to desensitize nociceptors in peripheraltissues, VR1 agonist vanilloid compounds have been used as topicalanesthetics. However, agonist application may itself cause burning pain,which limits this therapeutic use. Recently, it has been reported thatVR1 antagonists, including certain nonvanilloid compounds, are alsouseful for the treatment of pain (see, e.g., PCT InternationalApplication Publication Numbers WO 02/08221, WO 03/062209, WO 04/054582,WO 04/055003, WO 04/055004, WO 04/056774, WO 05/007646, WO 05/007648, WO05/007652, WO 05/009977, WO 05/009980, WO 05/009982, WO 05/049601, WO05/049613, WO 06/122200 and WO 06/120481).

Thus, compounds that interact with VR1, but do not elicit the initialpainful sensation of VR1 agonist vanilloid compounds, are desirable forthe treatment of chronic and acute pain, including neuropathic pain, aswell as other conditions that are responsive to capsaicin receptormodulation. The present invention fulfills this need, and providesfurther related advantages.

SUMMARY OF THE INVENTION

The present invention provides 2-phenoxy pyrimidinone analogues ofFormula A:

as well as pharmaceutically acceptable salts, solvates (e.g., hydrates)and esters of such compounds.

-   Within Formula A:

represents a fused 5- or 6-membered heteroaryl that contains 1, 2 or 3heteroatoms in the ring, said heteroatoms being independently chosenfrom O, N and S, with the remaining ring atoms being carbon, wherein thefused heteroaryl is optionally substituted; preferably the fusedheteroaryl is substituted with from 0 to 3, or from 0 to 2, substituentsindependently chosen from amino, hydroxy, C₁-C₆alkyl, C₁-C₆hydroxyalkyl,(C₃-C₇cycloalkyl)C₀-C₂alkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₂-C₆alkylether, C₁-C₆alkanoyloxy, C₁-C₆alkylsulfonylamino, C₁-C₆alkanonylamino,and mono- or di-(C₁-C₆alkyl)amino;

-   Ar is phenyl or a 5- or 6-membered heteroaryl, each of which is    optionally substituted, and each of which is preferably substituted    with from 0 to 4 or from 0 to 3 substituents that are independently    chosen from halogen, cyano, amino, nitro, C₁-C₆alkyl, C₂-C₆alkenyl,    C₂-C₆alkynyl, C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆alkoxy,    C₁-C₆haloalkoxy, (C₃-C₇cycloalkyl)C₀-C₄alkyl, and mono- or    di-(C₁-C₆alkyl)amino; and-   R₃ represents from 0 to 4, or from 0 to 3, substituents, which    substituents are preferably independently chosen from halogen,    hydroxy, cyano, amino, nitro, C₁-C₆alkyl, C₂-C₆alkenyl,    C₂-C₆alkynyl, C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆alkoxy,    C₁-C₆haloalkoxy, (C₃-C₇cycloalkyl)C₀-C₄alkyl, mono- or    di-(C₁-C₆alkyl)amino, and mono- or di-(C₁-C₆alkyl)aminosulfonyl.

The present invention further provides 2-phenoxy pyrimidinone analoguesof Formula I:

as well as pharmaceutically acceptable salts, solvates (e.g., hydrates)and esters of such compounds.

-   Within Formula I:

and R₃ are as described for Formula A;

-   X is N or CH that is optionally substituted with a substituent    represented by R₁; and-   R₁ represents from 0 to 3 substituents; which substituents are    preferably independently chosen from halogen, cyano, amino, nitro,    C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆haloalkyl,    C₁-C₆hydroxyalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,    (C₃-C₇cycloalkyl)C₀-C₄alkyl, and mono- or di-(C₁-C₆alkyl)amino.

Within certain aspects, compounds of Formula A and Formula I are VR1modulators and exhibit a K_(i) of no greater than 1 micromolar, 500nanomolar, 100 nanomolar, 50 nanomolar, 10 nanomolar or 1 nanomolar in acapsaicin receptor binding assay and/or have an EC₅₀ or IC₅₀ value of nogreater than 1 micromolar, 500 nanomolar, 100 nanomolar, 50 nanomolar,10 nanomolar or 1 nanomolar in an in vitro assay for determination ofcapsaicin receptor agonist or antagonist activity. In certainembodiments, such VR1 modulators are VR1 antagonists and exhibit nodetectable agonist activity in an in vitro assay of capsaicin receptoractivation (e.g., the assay provided in Example 6, herein) at aconcentration equal to the IC₅₀, 10 times the IC₅₀ or 100 times theIC₅₀.

Within certain aspects, compounds provided herein are labeled with adetectable marker (e.g., radiolabeled or fluorescein conjugated).

The present invention further provides, within other aspects,pharmaceutical compositions comprising at least one 2-phenoxypyrimidinone analogue in combination with a physiologically acceptablecarrier or excipient.

Within further aspects, methods are provided for reducing calciumconductance of a cellular capsaicin receptor, comprising contacting acell (e.g., neuronal, such as cells of the central nervous system orperipheral ganglia, urothelial or lung) that expresses a capsaicinreceptor with at least one VR1 modulator as described herein. Suchcontact may occur in vivo or in vitro and is generally performed using aconcentration of VR1 modulator that is sufficient to alter the bindingof vanilloid ligand to VR1 in vitro (using the assay provided in Example5) and/or VR1-mediated signal transduction (using an assay provided inExample 6).

Methods are further provided for inhibiting binding of vanilloid ligandto a capsaicin receptor. Within certain embodiments, the inhibitiontakes place in vitro. Such methods comprise contacting a capsaicinreceptor with at least one VR1 modulator as described herein, underconditions and in an amount or concentration sufficient to detectablyinhibit vanilloid ligand binding to the capsaicin receptor. Within otherembodiments, the capsaicin receptor is in a patient. Such methodscomprise contacting cells expressing a capsaicin receptor in a patientwith at least one VR1 modulator as described herein in an amount orconcentration that would be sufficient to detectably inhibit vanilloidligand binding to cells expressing a cloned capsaicin receptor in vitro.

The present invention further provides methods for treating a conditionresponsive to capsaicin receptor modulation in a patient, comprisingadministering to the patient a therapeutically effective amount of atleast one VR1 modulator as described herein.

Within other aspects, methods are provided for treating pain in apatient, comprising administering to a patient suffering from (or atrisk for) pain a therapeutically effective amount of at least one VR1modulator as described herein.

Methods are further provided for treating itch, urinary incontinence,overactive bladder, menopause symptoms, cough and/or hiccup in apatient, comprising administering to a patient suffering from (or atrisk for) one or more of the foregoing conditions a therapeuticallyeffective amount of at least one VR1 modulator as described herein.

Within other aspects, methods are provided for treating menopausesymptoms in a patient, comprising administering to a patient sufferingfrom (or at risk for) such symptoms a therapeutically effective amountof at least one VR1 modulator as described herein.

The present invention further provides methods for promoting weight lossin an obese patient, comprising administering to an obese patient atherapeutically effective amount of at least one VR1 modulator asdescribed herein.

Methods are further provided for identifying an agent that binds tocapsaicin receptor, comprising: (a) contacting capsaicin receptor with alabeled compound as described herein under conditions that permitbinding of the compound to capsaicin receptor, thereby generating bound,labeled compound; (b) detecting a signal that corresponds to the amountof bound, labeled compound in the absence of test agent; (c) contactingthe bound, labeled compound with a test agent; (d) detecting a signalthat corresponds to the amount of bound labeled compound in the presenceof test agent; and (e) detecting a decrease in signal detected in step(d), as compared to the signal detected in step (b).

Within further aspects, the present invention provides methods fordetermining the presence or absence of capsaicin receptor in a sample,comprising: (a) contacting a sample with a compound as described hereinunder conditions that permit binding of the compound to capsaicinreceptor; and (b) detecting a signal indicative of a level of thecompound bound to capsaicin receptor.

The present invention also provides packaged pharmaceuticalpreparations, comprising: (a) a pharmaceutical composition as describedherein in a container; and (b) instructions for using the composition totreat one or more conditions responsive to capsaicin receptormodulation, such as pain, itch, urinary incontinence, overactivebladder, menopause symptoms, cough, hiccup and/or obesity.

In yet another aspect, the present invention provides methods forpreparing the compounds disclosed herein, including the intermediates.

These and other aspects of the invention will become apparent uponreference to the following detailed description.

DETAILED DESCRIPTION

As noted above, the present invention provides 2-phenoxy pyrimidinoneanalogues. Such compounds may be used in vitro or in vivo, to modulatecapsaicin receptor activity in a variety of contexts.

Terminology

Compounds are generally described herein using standard nomenclature.For compounds having asymmetric centers, it should be understood that(unless otherwise specified) all of the optical isomers and mixturesthereof are encompassed. In addition, compounds with carbon-carbondouble bonds may occur in Z- and E-forms, with all isomeric forms of thecompounds being included in the present invention unless otherwisespecified. Where a compound exists in various tautomeric forms, arecited compound is not limited to any one specific tautomer, but ratheris intended to encompass all tautomeric forms. Certain compounds aredescribed herein using a general formula that includes variables (e.g.,R₁, A). Unless otherwise specified, each variable within such a formulais defined independently of any other variable, and any variable thatoccurs more than one time in a formula is defined independently at eachoccurrence.

The phrase “2-phenoxy pyrimidinone analogues,” as used herein,encompasses all compounds of Formula A, including those of Formula I, aswell as compounds of other Formulas provided herein (including anyenantiomers, racemates and stereoisomers) and pharmaceuticallyacceptable salts, solvates and esters of such compounds.

A “pharmaceutically acceptable salt” of a compound recited herein is anacid or base salt that is suitable for use in contact with the tissuesof human beings or animals without excessive toxicity orcarcinogenicity, and preferably without irritation, allergic response,or other problem or complication. Such salts include mineral and organicacid salts of basic residues such as amines, as well as alkali ororganic salts of acidic residues such as carboxylic acids. Specificpharmaceutically acceptable anions for use in salt formation include,but are not limited to, acetate, 2-acetoxybenzoate, ascorbate, benzoate,bicarbonate, bromide, calcium edetate, carbonate, chloride, citrate,dihydrochloride, diphosphate, ditartrate, edetate, estolate(ethylsuccinate), formate, fumarate, gluceptate, gluconate, glutamate,glycolate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroiodide, hydroxymaleate,hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate,maleate, mandelate, methylbromide, methylnitrate, methylsulfate, mucate,napsylate, nitrate, pamoate, pantothenate, phenylacetate, phosphate,polygalacturonate, propionate, salicylate, stearate, subacetate,succinate, sulfamate, sulfanilate, sulfate, sulfonates includingbesylate (benzenesulfonate), camsylate (camphorsulfonate), edisylate(ethane-1,2-disulfonate), esylate (ethanesulfonate),2-hydroxyethylsulfonate, mesylate (methanesulfonate), triflate(trifluoromethanesulfonate) and tosylate (p-toluenesulfonate), tannate,tartrate, teoclate and triethiodide. Similarly, pharmaceuticallyacceptable cations for use in salt formation include, but are notlimited to ammonium, benzathine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine, procaine, and metals such asaluminum, calcium, lithium, magnesium, potassium, sodium and zinc. Thoseof ordinary skill in the art will recognize further pharmaceuticallyacceptable salts for the compounds provided herein. In general, apharmaceutically acceptable acid or base salt can be synthesized from aparent compound that contains a basic or acidic moiety by anyconventional chemical method. Briefly, such salts can be prepared byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two; generally, the use ofnonaqueous media, such as ether, ethyl acetate, ethanol, methanol,isopropanol or acetonitrile, is preferred.

It will be apparent that each compound provided herein may, but neednot, be formulated as a solvate (e.g., hydrate) or non-covalent complex.In addition, the various crystal forms and polymorphs are within thescope of the present invention. Also provided herein are prodrugs of thecompounds of the recited Formulas. A “prodrug” is a compound that maynot fully satisfy the structural requirements of the compounds providedherein, but is modified in vivo, following administration to a patient,to produce a compound a formula provided herein. For example, a prodrugmay be an acylated derivative of a compound as provided herein. Prodrugsinclude compounds wherein hydroxy, amine or sulfhydryl groups are bondedto any group that, when administered to a mammalian subject, cleaves toform a free hydroxy, amino, or sulfhydryl group, respectively. Examplesof prodrugs include, but are not limited to, acetate, formate andbenzoate derivatives of alcohol and amine functional groups within thecompounds provided herein. Prodrugs of the compounds provided herein maybe prepared by modifying functional groups present in the compounds insuch a way that the modifications are cleaved in vivo to yield theparent compounds.

As used herein, the term “alkyl” refers to a straight or branched chainsaturated aliphatic hydrocarbon. Alkyl groups include groups having from1 to 8 carbon atoms (C₁-C₈alkyl), from 1 to 6 carbon atoms (C₁-C₆alkyl)and from 1 to 4 carbon atoms (C₁-C₄alkyl), such as methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl,isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methylpentyl.“C₀-C_(n)alkyl” refers to a single covalent bond (C₀) or an alkyl grouphaving from 1 to n carbon atoms; for example “C₀-C₄alkyl” refers to asingle covalent bond or a C₁-C₄alkyl group. In some instances, asubstituent of an alkyl group is specifically indicated. For example,“hydroxyalkyl” refers to an alkyl group substituted with at least onehydroxy substituent.

“Alkylene” refers to a divalent alkyl group, as defined above.C₁-C₂alkylene is methylene or ethylene; C₀-C₄alkylene is a singlecovalent bond or an alkylene group having 1, 2, 3 or carbon atoms;C₀-C₂alkylene is a single covalent bond or an alkylene group having 1 or2 carbon atoms.

“Alkenyl” refers to straight or branched chain alkene groups, whichcomprise at least one unsaturated carbon-carbon double bond. Alkenylgroups include C₂-C₈alkenyl, C₂-C₆alkenyl and C₂-C₄alkenyl groups, whichhave from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively, such asethenyl, allyl or isopropenyl. “Alkynyl” refers to straight or branchedchain alkyne groups, which have one or more unsaturated carbon-carbonbonds, at least one of which is a triple bond. Alkynyl groups includeC₂-C₈alkynyl, C₂-C₆alkynyl and C₂-C₄alkynyl groups, which have from 2 to8, 2 to 6 or 2 to 4 carbon atoms, respectively.

A “cycloalkyl” is a group that comprises one or more saturated and/orpartially saturated rings in which all ring members are carbon, such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, adamantyl, and partially saturated variants of theforegoing, such as cyclohexenyl. Cycloalkyl groups do not comprise anaromatic ring or a heterocyclic ring. Certain cycloalkyl groups areC₃-C₇cycloalkyl, in which the cycloalkyl group contains a single ringhaving from 3 to 7 ring members, all of which are carbon. A“(C₃-C₈cycloalkyl)C₀-C₄alkyl” is a C₃-C₈cycloalkyl group linked via asingle covalent bond or a C₁-C₄alkylene group.

By “alkoxy,” as used herein, is meant an alkyl group as described aboveattached via an oxygen bridge. Alkoxy groups include C₁-C₆alkoxy andC₁-C₄alkoxy groups, which have from 1 to 6 or from 1 to 4 carbon atoms,respectively. Methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy,neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy arerepresentative alkoxy groups.

Similarly, “alkylthio” refers to an alkyl group as described aboveattached via a sulfur bridge.

“Alkyl ether” refers to a linear or branched ether substituent (i.e., analkyl group that is substituted with an alkoxy group). Alkyl ethergroups include C₂-C₈alkyl ether, C₂-C₆alkyl ether and C₂-C₄alkyl ethergroups, which have 2 to 8, 6 or 4 carbon atoms, respectively. A C₂alkylether has the structure —CH₂—O—CH₃.

The term “alkanoyl” refers to an acyl group (e.g., —(C═O)-alkyl), inwhich carbon atoms are in a linear or branched alkyl arrangement andwhere attachment is through the carbon of the keto group. Alkanoylgroups have the indicated number of carbon atoms, with the carbon of theketo group being included in the numbered carbon atoms. For example aC₂alkanoyl group is an acetyl group having the formula —(C═O)CH₃;“C₁alkanoyl” refers to —(C═O)H. “C₁-C₆alkanoyl groups” contain from 1 to6 carbon atoms.

The term “alkanoyloxy” as used herein refers to an alkanoyl groupattached through an oxygen linker (i.e., a group having the generalstructure —O—C(═O)-alkyl). Alkanoyloxy groups include, for example,C₁-C₆alkanoyloxy groups, which have from one to six carbon atoms.

“Alkanoylamino,” as used herein, refers to an alkanoyl group attachedthrough an amino linker (i.e., a group having the general structure—N(R)—C(═O)-alkyl), in which R is hydrogen or C₁-C₆alkyl).

Alkanoylamino groups include, for example, C₁-C₆alkanoylamino groups,which have from 1 to 6 carbon atoms in the “alkyl” portion (i.e., thecarbon of the keto bridge is not included in the indicated number ofcarbon atoms).

“Alkylsulfonyl” refers to groups of the formula —(SO₂)-alkyl, in whichthe sulfur atom is the point of attachment. Alkylsulfonyl groups includeC₁-C₆alkylsulfonyl and C₁-C₄alkylsulfonyl groups, which have from 1 to 6or from 1 to 4 carbon atoms, respectively. Methylsulfonyl is onerepresentative alkylsulfonyl group.

“Alkylsulfonylamino” refers to an alkylsulfonyl group attached throughan amino linker (i.e., a group having the general structure—N(R)—(SO₂)-alkyl), in which R is hydrogen or C₁-C₆alkyl).Alkylsulfonylamino groups include, for example, C₁-C₆alkylsulfonylaminogroups, which have from 1 to 6 carbon atoms.

“Aminosulfonyl” refers to groups of the formula —(SO₂)—NH₂, in which thesulfur atom is the point of attachment. The term “mono- ordi-(C₁-C₆alkyl)aminosulfonyl” refers to groups that satisfy the formula—(SO₂)—NR₂, in which the sulfur atom is the point of attachment, and inwhich one R is C₁-C₆alkyl and the other R is hydrogen or anindependently chosen C₁-C₆alkyl.

“Alkylamino” refers to a secondary or tertiary amine that has thegeneral structure —NH-alkyl or —N(alkyl)(alkyl), wherein each alkyl isselected independently from alkyl, cycloalkyl and (cycloalkyl)alkylgroups. Such groups include, for example, mono- and di-(C₁-C₆alkyl)aminogroups, in which each C₁-C₆alkyl may be the same or different.

“Alkylaminoalkyl” refers to an alkylamino group linked via an alkylenegroup (i.e., a group having the general structure -alkylene-NH-alkyl or-alkylene-N(alkyl)(alkyl)) in which each alkyl is selected independentlyfrom alkyl, cycloalkyl and (cycloalkyl)alkyl groups. Alkylaminoalkylgroups include, for example, mono- and di-(C₁-C₈alkyl)aminoC₁-C₈alkyl,mono- and di-(C₁-C₆alkyl)aminoC₁-C₆alkyl and mono- anddi-(C₁-C₆alkyl)aminoC₁-C₄alkyl. “Mono- ordi-(C₁-C₆alkyl)aminoC₀-C₆alkyl” refers to a mono- ordi-(C₁-C₆alkyl)amino group linked via a single covalent bond or aC₁-C₆alkylene group. The following are representative alkylaminoalkylgroups:

It will be apparent that the definition of “alkyl” as used in the terms“alkylamino” and “alkylaminoalkyl” differs from the definition of“alkyl” used for all other alkyl-containing groups, in the inclusion ofcycloalkyl and (cycloalkyl)alkyl groups (e.g.,(C₃-C₇cycloalkyl)C₀-C₄alkyl).

The term “aminocarbonyl” refers to an amide group (i.e., —(C═O)NH₂). Theterm “mono- or di-(C₁-C₆alkyl)aminocarbonyl” refers to groups of theformula —(C═O)—N(R)₂, in which the carbonyl is the point of attachment,one R is C₁-C₆alkyl and the other R is hydrogen or an independentlychosen C₁-C₆alkyl.

The term “halogen” refers to fluorine, chlorine, bromine or iodine.

A “haloalkyl” is an alkyl group that is substituted with 1 or moreindependently chosen halogens (e.g., “C₁-C₆haloalkyl” groups have from 1to 6 carbon atoms). Examples of haloalkyl groups include, but are notlimited to, mono-, di- or tri-fluoromethyl; mono-, di- ortri-chloromethyl; mono-, di-, tri-, tetra- or penta-fluoroethyl; mono-,di-, tri-, tetra- or penta-chloroethyl; and1,2,2,2-tetrafluoro-1-trifluoromethyl-ethyl. Typical haloalkyl groupsare trifluoromethyl and difluoromethyl. The term “haloalkoxy” refers toa haloalkyl group as defined above that is linked via an oxygen bridge.

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —CONH₂ isattached through the carbon atom.

A “heteroaryl” is an aromatic group in which at least one aromatic ringcomprises at least one heteroatom selected from N, O and S. Heteroarylsinclude, for example, 5- and 6-membered heteroaryls such as imidazole,furan, furazan, isothiazole, isoxazole, oxadiazole, oxazole, pyrazine,pyrazole, pyridazine, pyridine, pyrimidine, tetrazole, thiazole andthiophene.

A “substituent,” as used herein, refers to a molecular moiety that iscovalently bonded to an atom within a molecule of interest. For example,a ring substituent may be a moiety such as a halogen, alkyl group,haloalkyl group or other group that is covalently bonded to an atom(preferably a carbon or nitrogen atom) that is a ring member.Substituents of aromatic groups are generally covalently bonded to aring carbon atom. The term “substitution” refers to replacing a hydrogenatom in a molecular structure with a substituent, such that the valenceon the designated atom is not exceeded, and such that a chemicallystable compound (i.e., a compound that can be isolated, characterized,and tested for biological activity) results from the substitution.

Groups that are “optionally substituted” are unsubstituted or aresubstituted by other than hydrogen at one or more available positions,typically 1, 2, 3, 4 or 5 positions, by one or more suitable groups(which may be the same or different). Optional substitution is alsoindicated by the phrase “substituted with from 0 to X substituents,”where X is the maximum number of possible substituents. Certainoptionally substituted groups are substituted with from 0 to 2, 3 or 4independently selected substituents (i.e., are unsubstituted orsubstituted with up to the recited maximum number of substituents).Other optionally substituted groups are substituted with at least onesubstituent (e.g., substituted with from 1 to 2, 3 or 4 independentlyselected substituents).

The terms “VR1” and “capsaicin receptor” are used interchangeably hereinto refer to a type 1 vanilloid receptor. Unless otherwise specified,these terms encompass both rat and human VR1 receptors (e.g., GenBankAccession Numbers AF327067, AJ277028 and NM_(—)018727; sequences ofcertain human VR1 cDNAs and the encoded amino acid sequences areprovided in U.S. Pat. No. 6,482,611), as well as homologues thereoffound in other species.

A “VR1 modulator,” also referred to herein as a “modulator,” is acompound that modulates VR1 activation and/or VR1-mediated signaltransduction. VR1 modulators specifically provided herein are compoundsof Formula A, and pharmaceutically acceptable salts, hydrates and estersthereof. Certain preferred VR1 modulators are not vanilloids. A VR1modulator may be a VR1 agonist or antagonist. Certain modulators bind toVR1 with a K_(i) that is less than 1 micromolar, preferably less than500 nanomolar, 100 nanomolar, 10 nanomolar or 1 nanomolar. Arepresentative assay for determining K_(i) at VR1 is provided in Example5, herein.

A modulator is considered an “antagonist” if it detectably inhibitsvanilloid ligand binding to VR1 and/or VR1-mediated signal transduction(using, for example, the representative assay provided in Example 6); ingeneral, such an antagonist inhibits VR1 activation with a IC₅₀ value ofless than 1 micromolar, preferably less than 500 nanomolar, and morepreferably less than 100 nanomolar, 10 nanomolar or 1 nanomolar withinthe assay provided in Example 6. VR1 antagonists include neutralantagonists and inverse agonists.

An “inverse agonist” of VR1 is a compound that reduces the activity ofVR1 below its basal activity level in the absence of added vanilloidligand. Inverse agonists of VR1 may also inhibit the activity ofvanilloid ligand at VR1 and/or binding of vanilloid ligand to VR1. Thebasal activity of VR1, as well as the reduction in VR1 activity due tothe presence of VR1 antagonist, may be determined from a calciummobilization assay, such as the assay of Example 6.

A “neutral antagonist” of VR1 is a compound that inhibits the activityof vanilloid ligand at VR1, but does not significantly change the basalactivity of the receptor (i.e., within a calcium mobilization assay asdescribed in Example 6 performed in the absence of vanilloid ligand, VR1activity is reduced by no more than 10%, preferably by no more than 5%,and more preferably by no more than 2%; most preferably, there is nodetectable reduction in activity). Neutral antagonists of VR1 mayinhibit the binding of vanilloid ligand to VR1.

As used herein a “capsaicin receptor agonist” or “VR1 agonist” is acompound that elevates the activity of the receptor above the basalactivity level of the receptor (i.e., enhances VR1 activation and/orVR1-mediated signal transduction). Capsaicin receptor agonist activitymay be identified using the representative assay provided in Example 6.In general, such an agonist has an EC₅₀ value of less than 1 micromolar,preferably less than 500 nanomolar, and more preferably less than 100nanomolar or 10 nanomolar within the assay provided in Example 6.

A “vanilloid” is any compound that comprises a phenyl ring with twooxygen atoms bound to adjacent ring carbon atoms (one of which carbonatom is located para to the point of attachment of a third moiety thatis bound to the phenyl ring). Capsaicin is a representative vanilloid. A“vanilloid ligand” is a vanilloid that binds to VR1 with a K_(i)(determined as described herein) that is no greater than 10 μM.Vanilloid ligand agonists include capsaicin, olvanil,N-arachidonoyl-dopamine and resiniferatoxin (RTX). Vanilloid ligandantagonists include capsazepine and iodo-resiniferatoxin.

A “therapeutically effective amount” (or dose) is an amount that, uponadministration to a patient, results in a discernible patient benefit(e.g., provides detectable relief from at least one condition beingtreated). Such relief may be detected using any appropriate criteria,including alleviation of one or more symptoms such as pain. Atherapeutically effective amount or dose generally results in aconcentration of compound in a body fluid (such as blood, plasma, serum,CSF, synovial fluid, lymph, cellular interstitial fluid, tears or urine)that is sufficient to alter the binding of vanilloid ligand to VR1 invitro (using the assay provided in Example 5) and/or VR1-mediated signaltransduction (using an assay provided in Example 6). It will be apparentthat the discernible patient benefit may be apparent afteradministration of a single dose, or may become apparent followingrepeated administration of the therapeutically effective dose accordingto a predetermined regimen, depending upon the indication for which thecompound is administered.

By “statistically significant,” as used herein, is meant results varyingfrom control at the p<0.1 level of significance as measured using astandard parametric assay of statistical significance such as astudent's T test.

A “patient” is any individual treated with a compound provided herein.Patients include humans, as well as other animals such as companionanimals (e.g., dogs and cats) and livestock. Patients may beexperiencing one or more symptoms of a condition responsive to capsaicinreceptor modulation (e.g., pain, exposure to vanilloid ligand, itch,urinary incontinence, overactive bladder, menopause symptoms,respiratory disorders, cough and/or hiccup), or may be free of suchsymptom(s) (i.e., treatment may be prophylactic in a patient consideredat risk for the development of such symptoms).

2-Phenoxy Pyrimidinone Analogues

As noted above, the present invention provides 2-phenoxy pyrimidinoneanalogues of Formula A. Within certain aspects, such compounds are VR1modulators that may be used in a variety of contexts, including in thetreatment of pain (e.g., neuropathic or peripheral nerve-mediated pain);exposure to capsaicin; exposure to acid, heat, light, tear gas, airpollutants (such as, for example, tobacco smoke), infectious agents(including viruses, bacteria and yeast), pepper spray or related agents;respiratory conditions such as asthma or chronic obstructive pulmonarydisease; itch; urinary incontinence or overactive bladder; menopausesymptoms; cough or hiccup; and/or obesity. Such compounds may also beused within in vitro assays (e.g., assays for receptor activity), asprobes for detection and localization of VR1 and as standards in ligandbinding and VR1-mediated signal transduction assays.

It has been found, within the context of the present invention, that the2-phenoxy pyrimidinone analogues provided herein exhibit an unexpectedlyhigh VR1-modulating activity due, at least in part, to the phenoxymoiety of Formula A and Formula I.

As noted above,

represents a fused, optionally substituted 5- or 6-membered heteroarylin which 1, 2 or 3 ring members are heteroatoms independently chosenfrom O, N and S, and the remaining ring members are carbon. Withincertain embodiments,

is substituted with from 0 to 2 substituents independently chosen fromC₁-C₆alkyl, (C₃-C₇cycloalkyl)C₀-C₂alkyl and C₁-C₆haloalkyl. Withinfurther embodiments,

is substituted with from 0 to 2 substituents independently chosen fromC₁-C₄alkyl, (C₃-C₅cycloalkyl)C₀-C₂alkyl and C₁-C₄haloalkyl.

Within certain embodiments,

is a 5-membered heteroaryl that is substituted with from 0 to 2substituents independently chosen from C₁-C₄alkyl,(C₃-C₅cycloalkyl)C₀-C₂alkyl and C₁-C₄haloalkyl. In other embodiments,

is a 5-membered heteroaryl represented by any of the formulae:

in which R′₄ is hydrogen, C₁-C₄alkyl, (C₃-C₅cycloalkyl)C₀-C₂alkyl,C₁-C₄haloalkyl, C₁-C₄hydroxyalkyl, C₁-C₄alkoxy, C₁-C₄alkanoylamino orC₁-C₄alkylsulfonylamino. Representative such groups include, forexample,

in which R₂ is, for example, hydrogen, cyano, aryl, heteroaryl, halogen,C₁-C₄alkyl, C₁-C₄haloalkyl or C₃-C₅cycloalkyl. Within certainembodiments,

is

It will be apparent that the orientation of such

moieties is intended to be retained as shown (e.g., if

is

then the bicyclic core

is

Within other embodiments,

is a 6-membered heteroaryl that is substituted with from 0 to 3substituents independently chosen from hydroxy, C₁-C₆alkyl,(C₃-C₇cycloalkyl)C₀-C₂alkyl, C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl,C₁-C₆alkoxy, mono-(C₁-C₆alkyl)amino, C₁-C₆alkanoylamino orC₁-C₆alkylsulfonylamino. Representative such groups include, forexample,

wherein R₄ represents from 0 to 3, preferably from 1 to 3, substituentsindependently chosen from hydroxy, C₁-C₄alkyl,(C₃-C₅cycloalkyl)C₀-C₂alkyl, C₁-C₄haloalkyl, C₁-C₄hydroxyalkyl,C₁-C₄alkoxy, mono-(C₁-C₄alkyl)amino, C₁-C₄alkanoylamino orC₁-C₄alkylsulfonylamino.

The variable R₁, within certain embodiments, represents from 0 to 3,preferably from 1 to 3, substituents independently chosen from halogen,cyano, C₁-C₄alkyl and C₁-C₄haloalkyl. For example, R₁ represents exactlyone substituent (e.g., at the para position of the ring Ar) withincertain such compounds. Within other such compounds, at least onesubstituent represented by R₁ is a halogen or CN; such substituent islocated at the para position of a 6-membered Ar moiety within certainsuch compounds. It will be apparent that, within Formula I, the paraposition refers to the position para to the point of attachment of theAr moiety to the pyrimidinone core; that is, the 4-position of thephenyl ring that results when X is CH, and the 6-position of thepyridin-3-yl ring that results when X is N.

Within certain embodiments, R₃ represents from 1 to 3 substituentsindependently chosen from halogen, cyano, C₁-C₄alkyl, C₁-C₄haloalkyl andC₁-C₄alkoxy.

In certain embodiments, compounds of Formula I further satisfy one ofFormulas II-VII:

in which R₂ is hydrogen, C₁-C₄alkyl, C₁-C₄haloalkyl or C₃-C₅cycloalkyl;R₃ represents from 1 to 3 substituents independently chosen fromhalogen, cyano, C₁-C₄alkyl, C₁-C₄haloalkyl and C₁-C₄alkoxy; R′₄ ishydrogen, C₁-C₄alkyl, (C₃-C₅cycloalkyl)C₀-C₂alkyl, C₁-C₄haloalkyl,C₁-C₄hydroxyalkyl, C₁-C₄alkoxy, C₁-C₄alkanoylamino orC₁-C₄alkylsulfonylamino; and R₅ is halogen or CN. In certain embodimentsof Formulas II-VII, R′₄ is H (i.e. such compounds further satisfy one ofFormulas III-VIIa:

in which variables are as described for Formulas II-VII. Within furtherembodiments, compounds of Formula I further satisfy Formula VIII or IX:

in which R₃ represents from 1 to 3 substituents independently chosenfrom halogen, cyano, C₁-C₄alkyl, C₁-C₄haloalkyl and C₁-C₄alkoxy; R₄represents from 0 to 2 substituents independently chosen from hydroxy,C₁-C₄alkyl, (C₃-C₅cycloalkyl)C₀-C₂alkyl, C₁-C₄haloalkyl,C₁-C₄hydroxyalkyl, C₁-C₄alkoxy, mono-(C₁-C₄alkyl)amino,C₁-C₄alkanoylamino or C₁-C₄alkylsulfonylamino; and R₅ is halogen or CN.

Representative 2-phenoxy pyrimidinone analogues and intermediatesprovided herein include, but are not limited to, those specificallydescribed in Examples 1-3. It will be apparent that the specificcompounds recited herein are representative only, and are not intendedto limit the scope of the present invention. Further, as noted above,all compounds of the present invention may be present as a free acid orbase, or as a pharmaceutically acceptable salt. In addition, other formssuch as hydrates and prodrugs of such compounds are specificallycontemplated by the present invention.

Within certain aspects of the present invention, 2-phenoxy pyrimidinoneanalogues provided herein detectably alter (modulate) VR1 activity, asdetermined using an in vitro VR1 functional assay such as a calciummobilization assay. As an initial screen for such activity, a VR1 ligandbinding assay may be used. References herein to a “VR1 ligand bindingassay” are intended to refer to a standard in vitro receptor bindingassay such as that provided in Example 5, and a “calcium mobilizationassay” (also referred to herein as a “signal transduction assay”) may beperformed as described in Example 6. Briefly, to assess binding to VR1,a competition assay may be performed in which a VR1 preparation isincubated with labeled (e.g., ¹²⁵I or ³H) compound that binds to VR1(e.g., a capsaicin receptor agonist such as RTX) and unlabeled testcompound. Within the assays provided herein, the VR1 used is preferablymammalian VR1, more preferably human or rat VR1. The receptor may berecombinantly expressed or naturally expressed. The VR1 preparation maybe, for example, a membrane preparation from HEK293 or CHO cells thatrecombinantly express human VR1. Incubation with a compound thatdetectably modulates vanilloid ligand binding to VR1 results in adecrease or increase in the amount of label bound to the VR1preparation, relative to the amount of label bound in the absence of thecompound. This decrease or increase may be used to determine the K_(i)at VR1 as described herein. In general, compounds that decrease theamount of label bound to the VR1 preparation within such an assay arepreferred.

Certain VR1 modulators provided herein detectably modulate VR1 activityat nanomolar (i.e., submicromolar) concentrations, at subnanomolarconcentrations, or at concentrations below 100 picomolar, 20 picomolar,10 picomolar or 5 picomolar.

As noted above, compounds that are VR1 antagonists are preferred withincertain embodiments. IC₅₀ values for such compounds may be determinedusing a standard in vitro VR1-mediated calcium mobilization assay, asprovided in Example 6. Briefly, cells expressing capsaicin receptor arecontacted with a compound of interest and with an indicator ofintracellular calcium concentration (e.g., a membrane permeable calciumsensitivity dye such as Fluo-3 or Fura-2 (Molecular Probes, Eugene,Oreg.), each of which produce a fluorescent signal when bound to Ca⁺⁺).Such contact is preferably carried out by one or more incubations of thecells in buffer or culture medium comprising either or both of thecompound and the indicator in solution. Contact is maintained for anamount of time sufficient to allow the dye to enter the cells (e.g., 1-2hours). Cells are washed or filtered to remove excess dye and are thencontacted with a vanilloid receptor agonist (e.g., capsaicin, RTX orolvanil), typically at a concentration equal to the EC₅₀ concentration,and a fluorescence response is measured. When agonist-contacted cellsare contacted with a compound that is a VR1 antagonist the fluorescenceresponse is generally reduced by at least 20%, preferably at least 50%and more preferably at least 80%, as compared to cells that arecontacted with the agonist in the absence of test compound. The IC₅₀ forVR1 antagonists provided herein is preferably less than 1 micromolar,less than 100 nM, less than 10 nM or less than 1 nM. In certainembodiments, VR1 antagonists provided herein exhibit no detectableagonist activity an in vitro assay of capsaicin receptor agonism at aconcentration of compound equal to the IC₅₀. Certain such antagonistsexhibit no detectable agonist activity an in vitro assay of capsaicinreceptor agonism at a concentration of compound that is 100-fold higherthan the IC₅₀.

In other embodiments, compounds that are capsaicin receptor agonists arepreferred. Capsaicin receptor agonist activity may generally bedetermined as described in Example 6. When cells are contacted with 1micromolar of a compound that is a VR1 agonist, the fluorescenceresponse is generally increased by an amount that is at least 30% of theincrease observed when cells are contacted with 100 nM capsaicin. TheEC₅₀ for VR1 agonists provided herein is preferably less than 1micromolar, less than 100 nM or less than 10 nM.

VR1 modulating activity may also, or alternatively, be assessed using acultured dorsal root ganglion assay as provided in Example 7 and/or anin vivo pain relief assay as provided in Example 8. VR1 modulatorsprovided herein preferably have a statistically significant specificeffect on VR1 activity within one or more functional assays providedherein.

Within certain embodiments, VR1 modulators provided herein do notsubstantially modulate ligand binding to other cell surface receptors,such as EGF receptor tyrosine kinase or the nicotinic acetylcholinereceptor. In other words, such modulators do not substantially inhibitactivity of a cell surface receptor such as the human epidermal growthfactor (EGF) receptor tyrosine kinase or the nicotinic acetylcholinereceptor (e.g., the IC₅₀ or IC₄₀ at such a receptor is preferablygreater than 1 micromolar, and most preferably greater than 10micromolar). Preferably, a modulator does not detectably inhibit EGFreceptor activity or nicotinic acetylcholine receptor activity at aconcentration of 0.5 micromolar, 1 micromolar or more preferably 10micromolar. Assays for determining cell surface receptor activity arecommercially available, and include the tyrosine kinase assay kitsavailable from Panvera (Madison, Wis.).

In certain embodiments, preferred VR1 modulators are non-sedating. Inother words, a dose of VR1 modulator that is twice the minimum dosesufficient to provide analgesia in an animal model for determining painrelief (such as a model provided in Example 8, herein) causes onlytransient (i.e., lasting for no more than ½ the time that pain relieflasts) or preferably no statistically significant sedation in an animalmodel assay of sedation (using the method described by Fitzgerald et al.(1988) Toxicology 49(2-3):433-9). Preferably, a dose that is five timesthe minimum dose sufficient to provide analgesia does not producestatistically significant sedation. More preferably, a VR1 modulatorprovided herein does not produce sedation at intravenous doses of lessthan 25 mg/kg (preferably less than 10 mg/kg) or at oral doses of lessthan 140 mg/kg (preferably less than 50 mg/kg, more preferably less than30 mg/kg).

If desired, compounds provided herein may be evaluated for certainpharmacological properties including, but not limited to, oralbioavailability (preferred compounds are orally bioavailable to anextent allowing for therapeutically effective concentrations of thecompound to be achieved at oral doses of less than 140 mg/kg, preferablyless than 50 mg/kg, more preferably less than 30 mg/kg, even morepreferably less than 10 mg/kg, still more preferably less than 1 mg/kgand most preferably less than 0.1 mg/kg), toxicity (a preferred compoundis nontoxic when a therapeutically effective amount is administered to asubject), side effects (a preferred compound produces side effectscomparable to placebo when a therapeutically effective amount of thecompound is administered to a subject), serum protein binding and invitro and in vivo half-life (a preferred compound exhibits an in vivohalf-life allowing for Q.I.D. dosing, preferably T.I.D. dosing, morepreferably B.I.D. dosing, and most preferably once-a-day dosing). Inaddition, differential penetration of the blood brain barrier may bedesirable for VR1 modulators used to treat pain by modulating CNS VR1activity such that total daily oral doses as described above providesuch modulation to a therapeutically effective extent, while low brainlevels of VR1 modulators used to treat peripheral nerve mediated painmay be preferred (i.e., such doses do not provide brain (e.g., CSF)levels of the compound sufficient to significantly modulate VR1activity). Routine assays that are well known in the art may be used toassess these properties, and identify superior compounds for aparticular use. For example, assays used to predict bioavailabilityinclude transport across human intestinal cell monolayers, includingCaco-2 cell monolayers. Penetration of the blood brain barrier of acompound in humans may be predicted from the brain levels of thecompound in laboratory animals given the compound (e.g., intravenously).Serum protein binding may be predicted from albumin binding assays.Compound half-life is inversely proportional to the frequency of dosageof a compound. In vitro half-lives of compounds may be predicted fromassays of microsomal half-life as described, for example, within Example7 of U.S. Patent Application Publication Number 2005/0070547.

As noted above, preferred compounds provided herein are nontoxic. Ingeneral, the term “nontoxic” shall be understood in a relative sense andis intended to refer to any substance that has been approved by theUnited States Food and Drug Administration (“FDA”) for administration tomammals (preferably humans) or, in keeping with established criteria, issusceptible to approval by the FDA for administration to mammals(preferably humans). In addition, a highly preferred nontoxic compoundgenerally satisfies one or more of the following criteria: (1) does notsubstantially inhibit cellular ATP production; (2) does notsignificantly prolong heart QT intervals; (3) does not cause substantialliver enlargement, or (4) does not cause substantial release of liverenzymes.

As used herein, a compound that does not substantially inhibit cellularATP production is a compound that satisfies the criteria set forth inExample 8 of U.S. Patent Application Publication Number 2005/0070547. Inother words, cells treated as described therein with 100 μM of such acompound exhibit ATP levels that are at least 50% of the ATP levelsdetected in untreated cells. In more highly preferred embodiments, suchcells exhibit ATP levels that are at least 80% of the ATP levelsdetected in untreated cells.

A compound that does not significantly prolong heart QT intervals is acompound that does not result in a statistically significantprolongation of heart QT intervals (as determined byelectrocardiography) in guinea pigs, minipigs or dogs uponadministration of a dose that yields a serum concentration equal to theEC₅₀ or IC₅₀ for the compound. In certain preferred embodiments, a doseof 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 40 or 50 mg/kg administeredparenterally or orally does not result in a statistically significantprolongation of heart QT intervals.

A compound does not cause substantial liver enlargement if dailytreatment of laboratory rodents (e.g., mice or rats) for 5-10 days witha dose that yields a serum concentration equal to the EC₅₀ or IC₅₀ forthe compound results in an increase in liver to body weight ratio thatis no more than 100% over matched controls. In more highly preferredembodiments, such doses do not cause liver enlargement of more than 75%or 50% over matched controls. If non-rodent mammals (e.g., dogs) areused, such doses should not result in an increase of liver to bodyweight ratio of more than 50%, preferably not more than 25%, and morepreferably not more than 10% over matched untreated controls. Preferreddoses within such assays include 0.01, 0.05. 0.1, 0.5, 1, 5, 10, 40 or50 mg/kg administered parenterally or orally.

Similarly, a compound does not promote substantial release of liverenzymes if administration of twice the minimum dose that yields a serumconcentration equal to the EC₅₀ or IC₅₀ at VR1 for the compound does notelevate serum levels of ALT, LDH or AST in laboratory animals (e.g.,rodents) by more than 100% over matched mock-treated controls. In morehighly preferred embodiments, such doses do not elevate such serumlevels by more than 75% or 50% over matched controls. Alternatively, acompound does not promote substantial release of liver enzymes if, in anin vitro hepatocyte assay, concentrations (in culture media or othersuch solutions that are contacted and incubated with hepatocytes invitro) that are equal to the EC₅₀ or IC₅₀ for the compound do not causedetectable release of any of such liver enzymes into culture mediumabove baseline levels seen in media from matched mock-treated controlcells. In more highly preferred embodiments, there is no detectablerelease of any of such liver enzymes into culture medium above baselinelevels when such compound concentrations are five-fold, and preferablyten-fold the EC₅₀ or IC₅₀ for the compound.

In other embodiments, certain preferred compounds do not inhibit orinduce microsomal cytochrome P450 enzyme activities, such as CYP1A2activity, CYP2A6 activity, CYP2C9 activity, CYP2C19 activity, CYP2D6activity, CYP2E1 activity or CYP3A4 activity at a concentration equal tothe EC₅₀ or IC₅₀ at VR1 for the compound.

Certain preferred compounds are not clastogenic (e.g., as determinedusing a mouse erythrocyte precursor cell micronucleus assay, an Amesmicronucleus assay, a spiral micronucleus assay or the like) at aconcentration equal the EC₅₀ or IC₅₀ for the compound. In otherembodiments, certain preferred compounds do not induce sister chromatidexchange (e.g., in Chinese hamster ovary cells) at such concentrations.

For detection purposes, as discussed in more detail below, VR1modulators provided herein may be isotopically-labeled or radiolabeled.For example, compounds may have one or more atoms replaced by an atom ofthe same element having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be present in the compounds provided herein include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F and³⁶Cl. In addition, substitution with heavy isotopes such as deuterium(i.e., ²H) can afford certain therapeutic advantages resulting fromgreater metabolic stability, for example increased in vivo half-life orreduced dosage requirements and, hence, may be preferred in somecircumstances.

Preparation of 2-Phenoxy Pyrimidinone Analogues

2-Phenoxy pyrimidinone analogues may generally be prepared usingstandard synthetic methods. Starting materials are commerciallyavailable from suppliers such as Sigma-Aldrich Corp. (St. Louis, Mo.),or may be synthesized from commercially available precursors usingestablished protocols. By way of example, a synthetic route similar tothat shown in any of the following Schemes may be used, together withsynthetic methods known in the art of synthetic organic chemistry. Eachvariable in the following schemes refers to any group consistent withthe description of the compounds provided herein.

Certain abbreviations used in the following Schemes and elsewhere hereininclude:

-   CDCl₃ deuterated chloroform-   δ chemical shift-   DCM dichloromethane-   DMAP 4-dimethylaminopyridine-   DMF dimethylformamide-   DMSO dimethylsulfoxide-   DPPF 1,1′-bis(diphenylphosphino)ferrocene-   Et ethyl-   EtOAc ethyl acetate-   EtOH ethanol-   h hour(s)-   ¹H NMR proton nuclear magnetic resonance-   HPLC high pressure liquid chromatography-   Hz hertz-   KO^(t)Bu potassium tert-butoxide-   min minute(s)-   MS mass spectrometry-   (M+1) mass+1-   Pd₂(dba)₃ tris(dibenzylidineacetone)dipalladium(0)-   RT room temperature-   TFA trifluoroacetic acid

In certain embodiments, a compound provided herein may contain one ormore asymmetric carbon atoms, so that the compound can exist indifferent stereoisomeric forms. Such forms can be, for example,racemates or optically active forms. As noted above, all stereoisomersare encompassed by the present invention. Nonetheless, it may bedesirable to obtain single enantiomers (i.e., optically active forms).Standard methods for preparing single enantiomers include asymmetricsynthesis and resolution of the racemates. Resolution of the racematescan be accomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent, or chromatographyusing, for example a chiral HPLC column.

Compounds may be radiolabeled by carrying out their synthesis usingprecursors comprising at least one atom that is a radioisotope. Eachradioisotope is preferably carbon (e.g., ¹⁴C), hydrogen (e.g., ³H),sulfur (e.g., ³⁵S), or iodine (e.g., ¹²⁵I). Tritium labeled compoundsmay also be prepared catalytically via platinum-catalyzed exchange intritiated acetic acid, acid-catalyzed exchange in tritiatedtrifluoroacetic acid, or heterogeneous-catalyzed exchange with tritiumgas using the compound as substrate. In addition, certain precursors maybe subjected to tritium-halogen exchange with tritium gas, tritium gasreduction of unsaturated bonds, or reduction using sodium borotritide,as appropriate. Preparation of radiolabeled compounds may beconveniently performed by a radioisotope supplier specializing in customsynthesis of radiolabeled probe compounds.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising one or more compounds provided herein, together with at leastone physiologically acceptable carrier or excipient. Pharmaceuticalcompositions may comprise, for example, one or more of water, buffers(e.g., neutral buffered saline or phosphate buffered saline), ethanol,mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, adjuvants,polypeptides or amino acids such as glycine, antioxidants, chelatingagents such as EDTA or glutathione and/or preservatives. In addition,other active ingredients may (but need not) be included in thepharmaceutical compositions provided herein.

Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal, rectalor parenteral administration. The term parenteral as used hereinincludes subcutaneous, intradermal, intravascular (e.g., intravenous),intramuscular, spinal, intracranial, intrathecal and intraperitonealinjection, as well as any similar injection or infusion technique. Incertain embodiments, compositions suitable for oral use are preferred.Such compositions include, for example, tablets, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsion,hard or soft capsules, or syrups or elixirs. Within yet otherembodiments, pharmaceutical compositions may be formulated as alyophilizate. Formulation for topical administration may be preferredfor certain conditions (e.g., in the treatment of skin conditions suchas burns or itch). Formulation for direct administration into thebladder (intravesicular administration) may be preferred for treatmentof urinary incontinence and overactive bladder.

Compositions intended for oral use may further comprise one or morecomponents such as sweetening agents, flavoring agents, coloring agentsand/or preserving agents in order to provide appealing and palatablepreparations. Tablets contain the active ingredient in admixture withphysiologically acceptable excipients that are suitable for themanufacture of tablets. Such excipients include, for example, inertdiluents (e.g., calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate), granulating and disintegrating agents(e.g., corn starch or alginic acid), binding agents (e.g., starch,gelatin or acacia) and lubricating agents (e.g., magnesium stearate,stearic acid or talc). Tablets may be formed using standard techniques,including dry granulation, direct compression and wet granulation. Thetablets may be uncoated or they may be coated by known techniques.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent(e.g., calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium (e.g., peanut oil, liquid paraffin or olive oil).

Aqueous suspensions contain the active material(s) in admixture withsuitable excipients, such as suspending agents (e.g., sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia);and dispersing or wetting agents (e.g., naturally-occurring phosphatidessuch as lecithin, condensation products of an alkylene oxide with fattyacids such as polyoxyethylene stearate, condensation products ofethylene oxide with long chain aliphatic alcohols such asheptadecaethyleneoxycetanol, condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides such as polyethylene sorbitan monooleate). Aqueoussuspensions may also comprise one or more preservatives, such as ethylor n-propyl p-hydroxybenzoate, one or more coloring agents, one or moreflavoring agents, and/or one or more sweetening agents, such as sucroseor saccharin.

Oily suspensions may be formulated by suspending the activeingredient(s) in a vegetable oil (e.g., arachis oil, olive oil, sesameoil or coconut oil) or in a mineral oil such as liquid paraffin. Theoily suspensions may contain a thickening agent such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and/or flavoring agents may be added to provide palatable oralpreparations. Such suspensions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, a suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, such as sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions may also be formulated as oil-in-wateremulsions. The oily phase may be a vegetable oil (e.g., olive oil orarachis oil), a mineral oil (e.g., liquid paraffin) or a mixturethereof. Suitable emulsifying agents include naturally-occurring gums(e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides(e.g., soy bean lecithin, and esters or partial esters derived fromfatty acids and hexitol), anhydrides (e.g., sorbitan monoleate) andcondensation products of partial esters derived from fatty acids andhexitol with ethylene oxide (e.g., polyoxyethylene sorbitan monoleate).An emulsion may also comprise one or more sweetening and/or flavoringagents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso comprise one or more demulcents, preservatives, flavoring agentsand/or coloring agents.

Formulations for topical administration typically comprise a topicalvehicle combined with active agent(s), with or without additionaloptional components. Suitable topical vehicles and additional componentsare well known in the art, and it will be apparent that the choice of avehicle will depend on the particular physical form and mode ofdelivery. Topical vehicles include water; organic solvents such asalcohols (e.g., ethanol or isopropyl alcohol) or glycerin; glycols(e.g., butylene, isoprene or propylene glycol); aliphatic alcohols(e.g., lanolin); mixtures of water and organic solvents and mixtures oforganic solvents such as alcohol and glycerin; lipid-based materialssuch as fatty acids, acylglycerols (including oils, such as mineral oil,and fats of natural or synthetic origin), phosphoglycerides,sphingolipids and waxes; protein-based materials such as collagen andgelatin; silicone-based materials (both non-volatile and volatile); andhydrocarbon-based materials such as microsponges and polymer matrices. Acomposition may further include one or more components adapted toimprove the stability or effectiveness of the applied formulation, suchas stabilizing agents, suspending agents, emulsifying agents, viscosityadjusters, gelling agents, preservatives, antioxidants, skin penetrationenhancers, moisturizers and sustained release materials. Examples ofsuch components are described in Martindale—The Extra Pharmacopoeia(Pharmaceutical Press, London 1993) and Remington: The Science andPractice of Pharmacy, 21^(st) ed., Lippincott Williams & Wilkins,Philadelphia, Pa. (2005). Formulations may comprise microcapsules, suchas hydroxymethylcellulose or gelatin-microcapsules, liposomes, albuminmicrospheres, microemulsions, nanoparticles or nanocapsules.

A topical formulation may be prepared in any of a variety of physicalforms including, for example, solids, pastes, creams, foams, lotions,gels, powders, aqueous liquids and emulsions. The physical appearanceand viscosity of such pharmaceutically acceptable forms can be governedby the presence and amount of emulsifier(s) and viscosity adjuster(s)present in the formulation. Solids are generally firm and non-pourableand commonly are formulated as bars or sticks, or in particulate form;solids can be opaque or transparent, and optionally can containsolvents, emulsifiers, moisturizers, emollients, fragrances,dyes/colorants, preservatives and other active ingredients that increaseor enhance the efficacy of the final product. Creams and lotions areoften similar to one another, differing mainly in their viscosity; bothlotions and creams may be opaque, translucent or clear and often containemulsifiers, solvents, and viscosity adjusting agents, as well asmoisturizers, emollients, fragrances, dyes/colorants, preservatives andother active ingredients that increase or enhance the efficacy of thefinal product. Gels can be prepared with a range of viscosities, fromthick or high viscosity to thin or low viscosity. These formulations,like those of lotions and creams, may also contain solvents,emulsifiers, moisturizers, emollients, fragrances, dyes/colorants,preservatives and other active ingredients that increase or enhance theefficacy of the final product. Liquids are thinner than creams, lotions,or gels and often do not contain emulsifiers. Liquid topical productsoften contain solvents, emulsifiers, moisturizers, emollients,fragrances, dyes/colorants, preservatives and other active ingredientsthat increase or enhance the efficacy of the final product.

Suitable emulsifiers for use in topical formulations include, but arenot limited to, ionic emulsifiers, cetearyl alcohol, non-ionicemulsifiers like polyoxyethylene oleyl ether, PEG-40 stearate,ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100stearate and glyceryl stearate. Suitable viscosity adjusting agentsinclude, but are not limited to, protective colloids or non-ionic gumssuch as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate,silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. Agel composition may be formed by the addition of a gelling agent such aschitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol,polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate.Suitable surfactants include, but are not limited to, nonionic,amphoteric, ionic and anionic surfactants. For example, one or more ofdimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60,polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleylbetaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammoniumlaureth sulfate may be used within topical formulations. Suitablepreservatives include, but are not limited to, antimicrobials such asmethylparaben, propylparaben, sorbic acid, benzoic acid, andformaldehyde, as well as physical stabilizers and antioxidants such asvitamin E, sodium ascorbate/ascorbic acid and propyl gallate. Suitablemoisturizers include, but are not limited to, lactic acid and otherhydroxy acids and their salts, glycerin, propylene glycol, and butyleneglycol. Suitable emollients include lanolin alcohol, lanolin, lanolinderivatives, cholesterol, petrolatum, isostearyl neopentanoate andmineral oils. Suitable fragrances and colors include, but are notlimited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitableadditional ingredients that may be included a topical formulationinclude, but are not limited to, abrasives, absorbents, anti-cakingagents, anti-foaming agents, anti-static agents, astringents (e.g.,witch hazel, alcohol and herbal extracts such as chamomile extract),binders/excipients, buffering agents, chelating agents, film formingagents, conditioning agents, propellants, opacifying agents, pHadjusters and protectants.

An example of a suitable topical vehicle for formulation of a gel is:hydroxypropylcellulose (2.1%); 70/30 isopropyl alcohol/water (90.9%);propylene glycol (5.1%); and Polysorbate 80 (1.9%). An example of asuitable topical vehicle for formulation as a foam is: cetyl alcohol(1.1%); stearyl alcohol (0.5%; Quaternium 52 (1.0%); propylene glycol(2.0%); ethanol 95 PGF3 (61.05%); deionized water (30.05%); P75hydrocarbon propellant (4.30%). All percents are by weight.

Typical modes of delivery for topical compositions include applicationusing the fingers; application using a physical applicator such as acloth, tissue, swab, stick or brush; spraying (including mist, aerosolor foam spraying); dropper application; sprinkling; soaking; andrinsing.

A pharmaceutical composition may be prepared as a sterile injectibleaqueous or oleaginous suspension. The compound(s) provided herein,depending on the vehicle and concentration used, can either be suspendedor dissolved in the vehicle. Such a composition may be formulatedaccording to the known art using suitable dispersing, wetting agentsand/or suspending agents such as those mentioned above. Among theacceptable vehicles and solvents that may be employed are water,1,3-butanediol, Ringer's solution and isotonic sodium chloride solution.In addition, sterile, fixed oils may be employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employed,including synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectible compositions,and adjuvants such as local anesthetics, preservatives and/or bufferingagents can be dissolved in the vehicle.

Pharmaceutical compositions may also be formulated as suppositories(e.g., for rectal administration). Such compositions can be prepared bymixing the drug with a suitable non-irritating excipient that is solidat ordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Suitable excipientsinclude, for example, cocoa butter and polyethylene glycols.

Compositions for inhalation typically can be provided in the form of asolution, suspension or emulsion that can be administered as a drypowder or in the form of an aerosol using a conventional propellant(e.g., dichlorodifluoromethane or trichlorofluoromethane).

Pharmaceutical compositions may be formulated for release at apre-determined rate. Instantaneous release may be achieved, for example,via sublingual administration (i.e., administration by mouth in such away that the active ingredient(s) are rapidly absorbed via the bloodvessels under the tongue rather than via the digestive tract).Controlled release formulations (i.e., formulations such as a capsule,tablet or coated tablet that slows and/or delays release of activeingredient(s) following administration) may be administered by, forexample, oral, rectal or subcutaneous implantation, or by implantationat a target site. In general, a controlled release formulation comprisesa matrix and/or coating that delays disintegration and absorption in thegastrointestinal tract (or implantation site) and thereby provides adelayed action or a sustained action over a longer period. One type ofcontrolled-release formulation is a sustained-release formulation, inwhich at least one active ingredient is continuously released over aperiod of time at a constant rate. Preferably, the therapeutic agent isreleased at such a rate that blood (e.g., plasma) concentrations aremaintained within the therapeutic range, but below toxic levels, over aperiod of time that is at least 4 hours, preferably at least 8 hours,and more preferably at least 12 hours. Such formulations may generallybe prepared using well known technology and administered by, forexample, oral, rectal or subcutaneous implantation, or by implantationat the desired target site. Carriers for use within such formulationsare biocompatible, and may also be biodegradable; preferably theformulation provides a relatively constant level of modulator release.The amount of modulator contained within a sustained release formulationdepends upon, for example, the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

Controlled release may be achieved by combining the active ingredient(s)with a matrix material that itself alters release rate and/or throughthe use of a controlled-release coating. The release rate can be variedusing methods well known in the art, including (a) varying the thicknessor composition of coating, (b) altering the amount or manner of additionof plasticizer in a coating, (c) including additional ingredients, suchas release-modifying agents, (d) altering the composition, particle sizeor particle shape of the matrix, and (e) providing one or morepassageways through the coating. The amount of modulator containedwithin a sustained release formulation depends upon, for example, themethod of administration (e.g., the site of implantation), the rate andexpected duration of release and the nature of the condition to betreated or prevented.

The matrix material, which itself may or may not serve acontrolled-release function, is generally any material that supports theactive ingredient(s). For example, a time delay material such asglyceryl monosterate or glyceryl distearate may be employed. Activeingredient(s) may be combined with matrix material prior to formation ofthe dosage form (e.g., a tablet). Alternatively, or in addition, activeingredient(s) may be coated on the surface of a particle, granule,sphere, microsphere, bead or pellet that comprises the matrix material.Such coating may be achieved by conventional means, such as bydissolving the active ingredient(s) in water or other suitable solventand spraying. Optionally, additional ingredients are added prior tocoating (e.g., to assist binding of the active ingredient(s) to thematrix material or to color the solution). The matrix may then be coatedwith a barrier agent prior to application of controlled-release coating.Multiple coated matrix units may, if desired, be encapsulated togenerate the final dosage form.

In certain embodiments, a controlled release is achieved through the useof a controlled release coating (i.e., a coating that permits release ofactive ingredient(s) at a controlled rate in aqueous medium). Thecontrolled release coating should be a strong, continuous film that issmooth, capable of supporting pigments and other additives, non-toxic,inert and tack-free. Coatings that regulate release of the modulatorinclude pH-independent coatings, pH-dependent coatings (which may beused to release modulator in the stomach) and enteric coatings (whichallow the formulation to pass intact through the stomach and into thesmall intestine, where the coating dissolves and the contents areabsorbed by the body). It will be apparent that multiple coatings may beemployed (e.g., to allow release of a portion of the dose in the stomachand a portion further along the gastrointestinal tract). For example, aportion of active ingredient(s) may be coated over an enteric coating,and thereby released in the stomach, while the remainder of activeingredient(s) in the matrix core is protected by the enteric coating andreleased further down the GI tract. pH dependent coatings include, forexample, shellac, cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropylmethylcellulose phthalate, methacrylic acidester copolymers and zein.

In certain embodiments, the coating is a hydrophobic material,preferably used in an amount effective to slow the hydration of thegelling agent following administration. Suitable hydrophobic materialsinclude alkyl celluloses (e.g., ethylcellulose orcarboxymethylcellulose), cellulose ethers, cellulose esters, acrylicpolymers (e.g., poly(acrylic acid), poly(methacrylic acid), acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, methacrylic acid alkamidecopolymer, poly(methyl methacrylate), polyacrylamide, ammoniomethacrylate copolymers, aminoalkyl methacrylate copolymer,poly(methacrylic acid anhydride) and glycidyl methacrylate copolymers)and mixtures of the foregoing. Representative aqueous dispersions ofethylcellulose include, for example, AQUACOAT® (FMC Corp., Philadelphia,Pa.) and SURELEASE® (Colorcon, Inc., West Point, Pa.), both of which canbe applied to the substrate according to the manufacturer'sinstructions. Representative acrylic polymers include, for example, thevarious EUDRAGIT® (Rohm America, Piscataway, N.J.) polymers, which maybe used singly or in combination depending on the desired releaseprofile, according to the manufacturer's instructions.

The physical properties of coatings that comprise an aqueous dispersionof a hydrophobic material may be improved by the addition or one or moreplasticizers. Suitable plasticizers for alkyl celluloses include, forexample, dibutyl sebacate, diethyl phthalate, triethyl citrate, tributylcitrate and triacetin. Suitable plasticizers for acrylic polymersinclude, for example, citric acid esters such as triethyl citrate andtributyl citrate, dibutyl phthalate, polyethylene glycols, propyleneglycol, diethyl phthalate, castor oil and triacetin.

Controlled-release coatings are generally applied using conventionaltechniques, such as by spraying in the form of an aqueous dispersion. Ifdesired, the coating may comprise pores or channels or to facilitaterelease of active ingredient. Pores and channels may be generated bywell known methods, including the addition of organic or inorganicmaterial that is dissolved, extracted or leached from the coating in theenvironment of use. Certain such pore-forming materials includehydrophilic polymers, such as hydroxyalkylcelluloses (e.g.,hydroxypropylmethylcellulose), cellulose ethers, synthetic water-solublepolymers (e.g., polyvinylpyrrolidone, cross-linked polyvinylpyrrolidoneand polyethylene oxide), water-soluble polydextrose, saccharides andpolysaccharides and alkali metal salts. Alternatively, or in addition, acontrolled release coating may include one or more orifices, which maybe formed my methods such as those described in U.S. Pat. Nos.3,845,770; 4,034,758; 4,077,407; 4,088,864; 4,783,337 and 5,071,607.Controlled-release may also be achieved through the use of transdermalpatches, using conventional technology (see, e.g., U.S. Pat. No.4,668,232).

Further examples of controlled release formulations, and componentsthereof, may be found, for example, in U.S. Pat. Nos. 5,524,060;4,572,833; 4,587,117; 4,606,909; 4,610,870; 4,684,516; 4,777,049;4,994,276; 4,996,058; 5,128,143; 5,202,128; 5,376,384; 5,384,133;5,445,829; 5,510,119; 5,618,560; 5,643,604; 5,891,474; 5,958,456;6,039,980; 6,143,353; 6,126,969; 6,156,342; 6,197,347; 6,387,394;6,399,096; 6,437,000; 6,447,796; 6,475,493; 6,491,950; 6,524,615;6,838,094; 6,905,709; 6,923,984; 6,923,988; and 6,911,217; each of whichis hereby incorporated by reference for its teaching of the preparationof controlled release dosage forms.

In addition to or together with the above modes of administration, acompound provided herein may be conveniently added to food or drinkingwater (e.g., for administration to non-human animals including companionanimals (such as dogs and cats) and livestock). Animal feed and drinkingwater compositions may be formulated so that the animal takes in anappropriate quantity of the composition along with its diet. It may alsobe convenient to present the composition as a premix for addition tofeed or drinking water.

Compounds are generally administered in a therapeutically effectiveamount. Preferred systemic doses are no higher than 50 mg per kilogramof body weight per day (e.g., ranging from about 0.001 mg to about 50 mgper kilogram of body weight per day), with oral doses generally beingabout 5-20 fold higher than intravenous doses (e.g., ranging from 0.01to 40 mg per kilogram of body weight per day).

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage unit will vary depending, forexample, upon the patient being treated, the particular mode ofadministration and any other co-administered drugs. Dosage unitsgenerally contain between from about 10 μg to about 500 mg of activeingredient. Optimal dosages may be established using routine testing,and procedures that are well known in the art.

Pharmaceutical compositions may be packaged for treating conditionsresponsive to VR1 modulation (e.g., treatment of exposure to vanilloidligand or other irritant, pain, itch, obesity or urinary incontinence).Packaged pharmaceutical compositions generally include (i) a containerholding a pharmaceutical composition that comprises at least one VR1modulator as described herein and (ii) instructions (e.g., labeling or apackage insert) indicating that the contained composition is to be usedfor treating a condition responsive to VR1 modulation in the patient.

Methods of Use

VR1 modulators provided herein may be used to alter activity and/oractivation of capsaicin receptors in a variety of contexts, both invitro and in vivo. Within certain aspects, VR1 antagonists may be usedto inhibit the binding of vanilloid ligand agonist (such as capsaicinand/or RTX) to capsaicin receptor in vitro or in vivo. In general, suchmethods comprise the step of contacting a capsaicin receptor with one ormore VR1 modulators provided herein, in the presence of vanilloid ligandin aqueous solution and under conditions otherwise suitable for bindingof the ligand to capsaicin receptor. The VR1 modulator(s) are generallypresent at a concentration that is sufficient to alter the binding ofvanilloid ligand to VR1 in vitro (using the assay provided in Example 5)and/or VR1-mediated signal transduction (using an assay provided inExample 6). The capsaicin receptor may be present in solution orsuspension (e.g., in an isolated membrane or cell preparation), or in acultured or isolated cell. Within certain embodiments, the capsaicinreceptor is expressed by a neuronal cell present in a patient, and theaqueous solution is a body fluid. Preferably, one or more VR1 modulatorsare administered to an animal in an amount such that the VR1 modulatoris present in at least one body fluid of the animal at a therapeuticallyeffective concentration that is 1 micromolar or less; preferably 500nanomolar or less; more preferably 100 nanomolar or less, 50 nanomolaror less, 20 nanomolar or less, or 10 nanomolar or less. For example,such compounds may be administered at a therapeutically effective dosethat is less than 20 mg/kg body weight, preferably less than 5 mg/kgand, in some instances, less than 1 mg/kg.

Also provided herein are methods for modulating, preferably reducing,the signal-transducing activity (i.e., the calcium conductance) of acellular capsaicin receptor. Such modulation may be achieved bycontacting a capsaicin receptor (either in vitro or in vivo) with one ormore VR1 modulators provided herein under conditions suitable forbinding of the modulator(s) to the receptor. The VR1 modulator(s) aregenerally present at a concentration that is sufficient to alter thebinding of vanilloid ligand to VR1 in vitro and/or VR1-mediated signaltransduction as described herein. The receptor may be present insolution or suspension, in a cultured or isolated cell preparation or ina cell within a patient. For example, the cell may be a neuronal cellthat is contacted in vivo in an animal. Alternatively, the cell may bean epithelial cell, such as a urinary bladder epithelial cell(urothelial cell) or an airway epithelial cell that is contacted in vivoin an animal. Modulation of signal tranducing activity may be assessedby detecting an effect on calcium ion conductance (also referred to ascalcium mobilization or flux). Modulation of signal transducing activitymay alternatively be assessed by detecting an alteration of a symptom(e.g., pain, burning sensation, broncho-constriction, inflammation,cough, hiccup, itch, menopause symptoms, urinary incontinence oroveractive bladder) of a patient being treated with one or more VR1modulators provided herein.

VR1 modulator(s) provided herein are preferably administered to apatient (e.g., a human) orally or topically, and are present within atleast one body fluid of the animal while modulating VR1signal-transducing activity. Preferred VR1 modulators for use in suchmethods modulate VR1 signal-transducing activity in vitro at aconcentration of 1 nanomolar or less, preferably 100 picomolar or less,more preferably 20 picomolar or less, and in vivo at a concentration of1 micromolar or less, 500 nanomolar or less, or 100 nanomolar or less ina body fluid such as blood.

The present invention further provides methods for treating conditionsresponsive to VR1 modulation. Within the context of the presentinvention, the term “treatment” encompasses both disease-modifyingtreatment and symptomatic treatment, either of which may be prophylactic(i.e., before the onset of symptoms, in order to prevent, delay orreduce the severity of symptoms) or therapeutic (i.e., after the onsetof symptoms, in order to reduce the severity and/or duration ofsymptoms). A condition is “responsive to VR1 modulation” if it ischaracterized by inappropriate activity of a capsaicin receptor,regardless of the amount of vanilloid ligand present locally, and/or ifmodulation of capsaicin receptor activity results in alleviation of thecondition or a symptom thereof. Such conditions include, for example,symptoms resulting from exposure to VR1-activating stimuli, pain,respiratory disorders (such as cough, asthma, chronic obstructivepulmonary disease, chronic bronchitis, cystic fibrosis and rhinitis,including allergic rhinitis, such as seasonal an perennial rhinitis, andnon-allergic rhinitis), depression, itch, menopause symptoms, urinaryincontinence, overactive bladder, acoustic injury (e.g., of thecochlea), tinnitus, hyperacusis, diabetes and prediabetic conditions(e.g., insulin resistance or glucose tolerance), hiccup and obesity, asdescribed in more detail below. Such conditions may be diagnosed andmonitored using criteria that have been established in the art. Patientsmay include humans, domesticated companion animals and livestock, withdosages as described above.

Treatment regimens may vary depending on the compound used and theparticular condition to be treated; however, for treatment of mostdisorders, a frequency of administration of 4 times daily or less ispreferred. In general, a dosage regimen of 2 times daily is morepreferred, with once a day dosing particularly preferred. For thetreatment of acute pain, a single dose that rapidly reaches effectiveconcentrations is desirable. It will be understood, however, that thespecific dose level and treatment regimen for any particular patientwill depend upon a variety of factors including the activity of thespecific compound employed, the age, body weight, general health, sex,diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy. In general, the use of the minimum dose sufficientto provide effective therapy is preferred. Patients may generally bemonitored for therapeutic effectiveness using medical or veterinarycriteria suitable for the condition being treated or prevented.

Patients experiencing symptoms resulting from exposure to capsaicinreceptor-activating stimuli include individuals with burns caused byheat, light, tear gas or acid and those whose mucous membranes areexposed (e.g., via ingestion, inhalation or eye contact) to capsaicin(e.g., from hot peppers or in pepper spray) or a related irritant suchas acid, tear gas, infectious agent(s) or air pollutant(s). Theresulting symptoms (which may be treated using VR1 modulators,especially antagonists, provided herein) may include, for example, pain,broncho-constriction and inflammation.

Pain that may be treated using the VR1 modulators provided herein may bechronic or acute and includes, but is not limited to, peripheralnerve-mediated pain (especially neuropathic pain). Compounds providedherein may be used in the treatment of, for example, postmastectomy painsyndrome, stump pain, phantom limb pain, oral neuropathic pain,toothache (dental pain), denture pain, postherpetic neuralgia, diabeticneuropathy, chemotherapy-induced neuropathy, reflex sympatheticdystrophy, trigeminal neuralgia, osteoarthritis, rheumatoid arthritis,fibromyalgia, Guillain-Barre syndrome, meralgia paresthetica,burning-mouth syndrome and/or pain associated with nerve and rootdamage, including as pain associated with peripheral nerve disorders(e.g., nerve entrapment and brachial plexus avulsions, amputation,peripheral neuropathies including bilateral peripheral neuropathy, ticdouloureux, atypical facial pain, nerve root damage, and arachnoiditis).Additional neuropathic pain conditions include causalgia (reflexsympathetic dystrophy—RSD, secondary to injury of a peripheral nerve),neuritis (including, for example, sciatic neuritis, peripheral neuritis,polyneuritis, optic neuritis, postfebrile neuritis, migrating neuritis,segmental neuritis and Gombault's neuritis), neuronitis, neuralgias(e.g., those mentioned above, cervicobrachial neuralgia, cranialneuralgia, geniculate neuralgia, glossopharyngial neuralgia, migranousneuralgia, idiopathic neuralgia, intercostals neuralgia, mammaryneuralgia, mandibular joint neuralgia, Morton's neuralgia, nasociliaryneuralgia, occipital neuralgia, red neuralgia, Sluder's neuralgia,splenopalatine neuralgia, supraorbital neuralgia and vidian neuralgia),surgery-related pain, musculoskeletal pain, myofascial pain syndromes,AIDS-related neuropathy, MS-related neuropathy, central nervous systempain (e.g., pain due to brain stem damage, sciatica, and ankylosingspondylitis), and spinal pain, including spinal cord injury-relatedpain. Headache, including headaches involving peripheral nerve activitymay also be treated as described herein. Such pain includes, forexample, such as sinus, cluster (i.e., migranous neuralgia) and tensionheadaches, migraine, temporomandibular pain and maxillary sinus pain.For example, migraine headaches may be prevented by administration of acompound provided herein as soon as a pre-migrainous aura is experiencedby the patient. Further conditions that can be treated as describedherein include Charcot's pains, intestinal gas pains, ear pain, heartpain, muscle pain, eye pain, orofacial pain (e.g., odontalgia),abdominal pain, gynaecological pain (e.g., menstrual pain,dysmenorrhoea, pain associated with cystitis, labor pain, chronic pelvicpain, chronic prostitis and endometriosis), acute and chronic back pain(e.g., lower back pain), gout, scar pain, hemorrhoidal pain, dyspepticpains, angina, nerve root pain, “non-painful” neuropathies, complexregional pain syndrome, homotopic pain and heterotopic pain—includingpain associated with carcinoma, often referred to as cancer pain (e.g.,in patients with bone cancer), pain (and inflammation) associated withvenom exposure (e.g., due to snake bite, spider bite, or insect sting)and trauma associated pain (e.g., post-surgical pain, episiotomy pain,pain from cuts, musculoskeletal pain, bruises and broken bones, and burnpain, especially primary hyperalgesia associated therewith). Additionalpain conditions that may be treated as described herein include painassociated with respiratory disorders as described above, autoimmunediseases, immunodeficiency disorders, hot flashes, inflammatory boweldisease, gastroesophageal reflux disease (GERD), irritable bowelsyndrome and/or inflammatory bowel disease.

Within certain aspects, VR1 modulators provided herein may be used forthe treatment of mechanical pain. As used herein, the term “mechanicalpain” refers to pain other than headache pain that is not neuropathic ora result of exposure to heat, cold or external chemical stimuli.Mechanical pain includes physical trauma (other than thermal or chemicalburns or other irritating and/or painful exposures to noxious chemicals)such as post-surgical pain and pain from cuts, bruises and broken bones;toothache; denture pain; nerve root pain; osteoarthritis; rheumatoidarthritis; fibromyalgia; meralgia paresthetica; back pain;cancer-associated pain; angina; carpel tunnel syndrome; and painresulting from bone fracture, labor, hemorrhoids, intestinal gas,dyspepsia, and menstruation.

Itching conditions that may be treated include psoriatic pruritus, itchdue to hemodialysis, aguagenic pruritus, and itching associated withvulvar vestibulitis, contact dermatitis, insect bites and skinallergies. Urinary tract conditions that may be treated as describedherein include urinary incontinence (including overflow incontinence,urge incontinence and stress incontinence), as well as overactive orunstable bladder conditions (including bladder detrusor hyper-reflexia,detrusor hyper-reflexia of spinal origin and bladder hypersensitivity).In certain such treatment methods, VR1 modulator is administered via acatheter or similar device, resulting in direct injection of VR1modulator into the bladder. Compounds provided herein may also be usedas anti-tussive agents (to prevent, relieve or suppress coughing,including cough induced by medications such as ACE inhibitors) and forthe treatment of hiccup, for the treatment of menopause symptoms such ashot flashes, and to promote weight loss in an obese patient.

Within other aspects, VR1 modulators provided herein may be used withincombination therapy for the treatment of conditions involving painand/or inflammatory components. Such conditions include, for example,autoimmune disorders and pathologic autoimmune responses known to havean inflammatory component including, but not limited to, arthritis(especially rheumatoid arthritis), psoriasis, Crohn's disease, lupuserythematosus, irritable bowel syndrome, tissue graft rejection, andhyperacute rejection of transplanted organs. Other such conditionsinclude trauma (e.g., injury to the head or spinal cord), cardio- andcerebro-vascular disease and certain infectious diseases.

Suitable dosages for VR1 modulator within such combination therapy aregenerally as described above. Dosages and methods of administration ofanti-inflammatory agents can be found, for example, in themanufacturer's instructions in the Physician's Desk Reference. Incertain embodiments, the combination administration of a VR1 modulatorwith an anti-inflammatory agent results in a reduction of the dosage ofthe anti-inflammatory agent required to produce a therapeutic effect(i.e., a decrease in the minimum therapeutically effective amount).Thus, preferably, the dosage of anti-inflammatory agent in a combinationor combination treatment method is less than the maximum dose advised bythe manufacturer for administration of the anti-inflammatory agentwithout combination administration of a VR1 antagonist. More preferablythis dosage is less than ¾, even more preferably less than ½, and highlypreferably, less than ¼ of the maximum dose, while most preferably thedose is less than 10% of the maximum dose advised by the manufacturerfor administration of the anti-inflammatory agent(s) when administeredwithout combination administration of a VR1 antagonist. It will beapparent that the dosage amount of VR1 antagonist component of thecombination needed to achieve the desired effect may similarly beaffected by the dosage amount and potency of the anti-inflammatory agentcomponent of the combination.

In certain preferred embodiments, the combination administration of aVR1 modulator with an anti-inflammatory agent is accomplished bypackaging one or more VR1 modulators and one or more anti-inflammatoryagents in the same package, either in separate containers within thepackage or in the same contained as a mixture of one or more VR1antagonists and one or more anti-inflammatory agents. Preferred mixturesare formulated for oral administration (e.g., as pills, capsules,tablets or the like). In certain embodiments, the package comprises alabel bearing indicia indicating that the one or more VR1 modulators andone or more anti-inflammatory agents are to be taken together for thetreatment of an inflammatory pain condition.

Within further aspects, VR1 modulators provided herein may be used incombination with one or more additional pain relief medications. Certainsuch medications are also anti-inflammatory agents, and are listedabove. Other such medications are analgesic agents, including narcoticagents which typically act at one or more opioid receptor subtypes(e.g., μ, κ and/or δ), preferably as agonists or partial agonists. Suchagents include opiates, opiate derivatives and opioids, as well aspharmaceutically acceptable salts and hydrates thereof. Specificexamples of narcotic analgesics include, within preferred embodiments,alfentanil, alphaprodine, anileridine, bezitramide, buprenorphine,butorphanol, codeine, diacetyldihydromorphine, diacetylmorphine,dihydrocodeine, diphenoxylate, ethylmorphine, fentanyl, heroin,hydrocodone, hydromorphone, isomethadone, levomethorphan, levorphane,levorphanol, meperidine, metazocine, methadone, methorphan, metopon,morphine, nalbuphine, opium extracts, opium fluid extracts, powderedopium, granulated opium, raw opium, tincture of opium, oxycodone,oxymorphone, paregoric, pentazocine, pethidine, phenazocine, piminodine,propoxyphene, racemethorphan, racemorphan, sulfentanyl, thebaine andpharmaceutically acceptable salts and hydrates of the foregoing agents.

Other examples of narcotic analgesic agents include acetorphine,acetyldihydrocodeine, acetylmethadol, allylprodine, alphracetylmethadol,alphameprodine, alphamethadol, benzethidine, benzylmorphine,betacetylmethadol, betameprodine, betamethadol, betaprodine,clonitazene, codeine methylbromide, codeine-N-oxide, cyprenorphine,desomorphine, dextromoramide, diampromide, diethylthiambutene,dihydromorphine, dimenoxadol, dimepheptanol, dimethylthiamubutene,dioxaphetyl butyrate, dipipanone, drotebanol, ethanol,ethylmethylthiambutene, etonitazene, etorphine, etoxeridine,furethidine, hydromorphinol, hydroxypethidine, ketobemidone,levomoramide, levophenacylmorphan, methyldesorphine,methyldihydromorphine, morpheridine, morphine methylpromide, morphinemethylsulfonate, morphine-N-oxide, myrophin, naloxone, naltyhexone,nicocodeine, nicomorphine, noracymethadol, norlevorphanol, normethadone,normorphine, norpipanone, pentazocaine, phenadoxone, phenampromide,phenomorphan, phenoperidine, piritramide, pholcodine, proheptazoine,properidine, propiran, racemoramide, thebacon, trimeperidine and thepharmaceutically acceptable salts and hydrates thereof.

Further specific representative analgesic agents include, for exampleacetaminophen (paracetamol); ibuprofen; aspirin and other NSAIDsdescribed above; NR2B antagonists; bradykinin antagonists; anti-migraineagents; anticonvulsants such as oxcarbazepine and carbamazepine;antidepressants (such as TCAs, SSRIs, SNRIs, substance P antagonists,etc.); spinal blocks; pentazocine/naloxone; meperidine; levorphanol;buprenorphine; hydromorphone; fentanyl; sufentanyl; oxycodone;oxycodone/acetaminophen, nalbuphine and oxymorphone. Still furtheranalgesic agents include CB2-receptor agonists, such as AM1241,capsaicin receptor antagonists and compounds that bind to the α2δsubunit of voltage-gated calcium channels, such as gabapentin andpregabalin.

Representative anti-migraine agents for use in combination with a VR1modulator provided herein include CGRP antagonists, ergotamines and5-HT₁ agonists, such as sumatripan, naratriptan, zolmatriptan andrizatriptan.

Within still further aspects, modulators provided herein may be used,for example, in the treatment of pulmonary disorders such as asthma, incombination with one or more beta(2)-adrenergic receptor agonists orleukotriene receptor antagonists (e.g., agents that inhibits thecysteinyl leukotriene CysLT₁ receptor). CysLT₁ antagonists includemontelukast, zafirlukast, and pranlukast.

For the treatment or prevention of cough, a VR1 modulator as providedherein may be used in combination with other medication designed totreat this condition, such as antibiotics, anti-inflammatory agents,cystinyl leukotrienes, histamine antagonists, corticosteroids, opioids,NMDA antagonists, proton pump inhibitors, nociceptin, neurokinin (NK1,NK2 and NK3) and bradykinin (BK1 and BK2) receptor antagonists,cannabinoids, blockers of Na+-dependent channels and large conductanceCa⁺²-dependent K⁺-channel activators. Specific agents includedexbrompheniramine plus pseudoephedrine, loratadine, oxymetazoline,ipratropium, albuterol, beclomethasone, morphine, codeine, pholcodeineand dextromethorphan.

The present invention further provides combination therapy for thetreatment of urinary incontinence. Within such aspects, a VR1 modulatorprovided herein may be used in combination with other medicationdesigned to treat this condition, such as estrogen replacement therapy,progesterone congeners, electrical stimulation, calcium channelblockers, antispasmodic agents, cholinergic antagonists, antimuscarinicdrugs, tricyclic antidepressants, SNRIs, beta adrenoceptor agonists,phosphodiesterase inhibitors, potassium channel openers,nociceptin/orphanin FQ (OP4) agonists, neurokinin (NK1 and NK2)antagonists, P2X3 antagonists, musculotrophic drugs and sacralneuromodulation. Specific agents include oxybutinin, emepronium,tolterodine, flavoxate, flurbiprofen, tolterodine, dicyclomine,propiverine, propantheline, dicyclomine, imipramine, doxepin,duloxetine, 1-deamino-8-D-arginine vasopressin, muscarinic receptorantagonists such as tolterodine and anticholinergic agents such asoxybutynin.

Suitable dosages for VR1 modulator within such combination therapy aregenerally as described above. Dosages and methods of administration ofother pain relief medications can be found, for example, in themanufacturer's instructions in the Physician's Desk Reference. Incertain embodiments, the combination administration of a VR1 modulatorwith one or more additional pain medications results in a reduction ofthe dosage of each therapeutic agent required to produce a therapeuticeffect (e.g., the dosage or one or both agent may less than ¾, less than½, less than ¼ or less than 10% of the maximum dose listed above oradvised by the manufacturer).

For use in combination therapy, pharmaceutical compositions as describedabove may further comprise one or more additional medications asdescribed above. In certain such compositions, the additional medicationis an analgesic. Also provided herein are packaged pharmaceuticalpreparations comprising one or more VR1 modulators and one or moreadditional medications (e.g., analgesics) in the same package. Suchpackaged pharmaceutical preparations generally include (i) a containerholding a pharmaceutical composition that comprises at least one VR1modulator as described herein; (ii) a container holding a pharmaceuticalcomposition that comprises at least one additional medication (such as apain relief and/or anti-inflammatory medication) as described above and(iii) instructions (e.g., labeling or a package insert) indicating thatthe compositions are to be used simultaneously, separately orsequentially for treating or preventing a condition responsive to VR1modulation in the patient (such as a condition in which pain and/orinflammation predominates).

Compounds that are VR1 agonists may further be used, for example, incrowd control (as a substitute for tear gas) or personal protection(e.g., in a spray formulation) or as pharmaceutical agents for thetreatment of pain, itch, menopause symptoms, urinary incontinence oroveractive bladder via capsaicin receptor desensitization. In general,compounds for use in crowd control or personal protection are formulatedand used according to conventional tear gas or pepper spray technology.

Within separate aspects, the present invention provides a variety ofnon-pharmaceutical in vitro and in vivo uses for the compounds providedherein. For example, such compounds may be labeled and used as probesfor the detection and localization of capsaicin receptor (in samplessuch as cell preparations or tissue sections, preparations or fractionsthereof). In addition, compounds provided herein that comprise asuitable reactive group (such as an aryl carbonyl, nitro or azide group)may be used in photoaffinity labeling studies of receptor binding sites.In addition, compounds provided herein may be used as positive controlsin assays for receptor activity, as standards for determining theability of a candidate agent to bind to capsaicin receptor, or asradiotracers for positron emission tomography (PET) imaging or forsingle photon emission computerized tomography (SPECT). Such methods canbe used to characterize capsaicin receptors in living subjects. Forexample, a VR1 modulator may be labeled using any of a variety of wellknown techniques (e.g., radiolabeled with a radionuclide such astritium, as described herein), and incubated with a sample for asuitable incubation time (e.g., determined by first assaying a timecourse of binding). Following incubation, unbound compound is removed(e.g., by washing), and bound compound detected using any methodsuitable for the label employed (e.g., autoradiography or scintillationcounting for radiolabeled compounds; spectroscopic methods may be usedto detect luminescent groups and fluorescent groups). As a control, amatched sample containing labeled compound and a greater (e.g., 10-foldgreater) amount of unlabeled compound may be processed in the samemanner. A greater amount of detectable label remaining in the testsample than in the control indicates the presence of capsaicin receptorin the sample. Detection assays, including receptor autoradiography(receptor mapping) of capsaicin receptor in cultured cells or tissuesamples may be performed as described by Kuhar in sections 8.1.1 to8.1.9 of Current Protocols in Pharmacology (1998) John Wiley & Sons, NewYork.

Compounds provided herein may also be used within a variety of wellknown cell separation methods. For example, modulators may be linked tothe interior surface of a tissue culture plate or other support, for useas affinity ligands for immobilizing and thereby isolating, capsaicinreceptors (e.g., isolating receptor-expressing cells) in vitro. Withinone preferred embodiment, a modulator linked to a fluorescent marker,such as fluorescein, is contacted with the cells, which are thenanalyzed (or isolated) by fluorescence activated cell sorting (FACS).

VR1 modulators provided herein may further be used within assays for theidentification of other agents that bind to capsaicin receptor. Ingeneral, such assays are standard competition binding assays, in whichbound, labeled VR1 modulator is displaced by a test compound. Briefly,such assays are performed by: (a) contacting capsaicin receptor with aradiolabeled VR1 modulator as described herein, under conditions thatpermit binding of the VR1 modulator to capsaicin receptor, therebygenerating bound, labeled VR1 modulator; (b) detecting a signal thatcorresponds to the amount of bound, labeled VR1 modulator in the absenceof test agent; (c) contacting the bound, labeled VR1 modulator with atest agent; (d) detecting a signal that corresponds to the amount ofbound labeled VR1 modulator in the presence of test agent; and (e)detecting a decrease in signal detected in step (d), as compared to thesignal detected in step (b).

The following Examples are offered by way of illustration and not by wayof limitation. Unless otherwise specified all reagents and solvent areof standard commercial grade and are used without further purification.Using routine modifications, the starting materials may be varied andadditional steps employed to produce other compounds provided herein.

EXAMPLES

Mass Spectroscopy data provide in the following examples is ElectrosprayMS, obtained in positive ion mode with a 15V or 30V cone voltage, usinga Micromass Time-of-Flight LCT, equipped with a Waters 600 pump, Waters996 photodiode array detector, Gilson 215 autosampler, and a Gilson 841microinjector. MassLynx (Advanced Chemistry Development, Inc; Toronto,Canada) version 4.0 software is used for data collection and analysis.Sample volume of 1 microliter is injected onto a 50×4.6 mm ChromolithSpeedROD C18 column, and eluted using a 2-phase linear gradient at 6ml/min flow rate. Sample is detected using total absorbance count overthe 220-340 nm UV range. The elution conditions are: Mobile PhaseA-95/5/0.05 Water/Methanol/TFA; Mobile Phase B-5/95/0.025Water/Methanol/TFA.

Gradient: Time (min) % B 0 10 0.5 100 1.2 100 1.21 10The total run time is 2 minutes inject to inject.

Example 1 Preparation of Representative Intermediates

This Example illustrates the preparation of representative intermediatesuseful in the synthesis of 2-phenoxy pyrimidinone derivatives.

A. Ethyl 3-nitriloalaninate

A mixture of ethyl cyanoglyoxylate-2-oxime (50 g, 352 mmol) in 440 mL ofwater is cautiously treated with 340 mL of saturated aqueous NaHCO₃,followed by portionwise addition of sodium hydrosulfite (165 g, 950mmol). The reaction is then heated to an internal temperature of 35° C.for 35 min. After cooling to RT, the reaction is saturated with NaCl(approx. 250 g) and extracted with CH₂Cl₂ (6×150 mL). The combinedCH₂Cl₂ extracts are dried (Na₂SO₄), filtered, and concentrated in vacuoto give the title compound as a brown oil. ¹H NMR (400 MHz, CDCl₃) δ4.43 (1H, s), 4.34 (2H, q, J 7.2), 2.30 (2H, bs), 1.35 (3H, t, J 7.2).

B. Ethyl 5-amino-1-methyl-1H-imidazole-4-carboxylate

A mixture of ethyl 3-nitriloalaninate (26.7 g, 208 mmol) and triethylorthoformate (34.6 mL, 208 mmol) in 340 mL of acetonitrile is heated to90° C. and stirred for 70 min. After cooling to RT, methylamine (25.9 mLof a 33 wt % solution in EtOH, 208 mmol) is added, followed by 20 h ofstirring at RT. The reaction mixture is then concentrated in vacuo anddissolved in approx. 200 mL of 1N HCl. The aqueous solution is washedwith CH₂Cl₂ (3×100 mL), basified to pH=9-10 with solid NaHCO₃, andextracted with CH₂Cl₂ (5×100 mL). The combined CH₂Cl₂ extracts are dried(Na₂SO₄), filtered, and concentrated to give a brown solid. The solid isslurried in EtOAc, filtered, and washed with Et₂O to give the titlecompound as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.03 (1H, s),4.88 (2H, bs), 4.33 (2H, q, J 7.2), 3.45 (3H, s), 1.37 (3H, t, J 7.2).

C.1-(4-Fluorophenyl)-9-methyl-2-thioxo-1,2,3,9-tetrahydro-6H-purin-6-onehydrochloride

Ethyl 5-amino-1-methyl-1H-imidazole-4-carboxylate (8.45 g, 0.05 moles)and 4-fluorophenyl isothiocyanate (7.65 g, 0.05 moles) are stirred inpyridine (125 mL) at 45° C. for 20 h. The reaction mixture isconcentrated under vacuum and diluted by the addition of ice cold water.The reaction mixture is extracted with CH₂Cl₂ (2×250 mL), washed withwater (200 mL) and dried over MgSO₄. The filtrate is evaporated in vacuoto give crude intermediate as red orange viscous oil. The oil isslurried in 1% aqueous sodium hydroxide solution (300 mL) and heated at90° C. for 20 h. The reaction mixture is cooled and the solid isfiltered. The filtrate is evaporated in vacuo to reduced volume (100mL). The mixture is acidified using concentrated HCl to pH 4.0 andallowed stand at RT overnight. The yellow solid which separates isfiltered and dried at 70° C., to afford the title compound. ¹H NMR (400MHz, DMSO-d₆) δ 7.8 (1 H, s), 7.2-7.4 (4H, m), 3.74 (3H, s).

D. 2-Chloro-1-(4-fluorophenyl)-9-ethyl-1,9-dihydro-6H-purin-6-one

1-(4-Fluorophenyl)-9-methyl-2-thioxo-1,2,3,9-tetrahydro-6H-purin-6-onehydrochloride (6.5 g, 0.021 mol) is suspended in a large excess ofphosphorous oxychloride (150 mL) and heated to 135° C. for 40 h. Thereaction mixture is cooled, evaporated in vacuo, and azeotroped twicewith toluene. The resulting sticky brown oil is dissolved in DCM (200mL) then neutralized with saturated NaHCO₃ (aqueous). The aqueous layeris extracted with DCM (2×200 mL) and dried (MgSO₄). The dried extract isfiltered and concentrated under vacuum to afford crude product as alight brown solid. The crude product is purified by flash columnchromatography using 1-2.5% MeOH/CH₂Cl₂ to afford the title compound aswhite solid. ¹H NMR (400 MHz, CDCl₃) δ 7.75 (1H, s), 7.2-7.35 (4H, m),3.85 (3H, s).

E. Ethyl 3-aminopyridine-2-carboxylate

A mixture of 3-aminopyridine-2-carboxylic acid (6.4 g, 46.3 mmol) in 26mL of EtOH and 8 mL of concentrated sulfuric acid is heated to refluxfor 2 days. After cooling, the mixture is concentrated to about 15-20 mLand poured into 20 g of ice. The mixture is basified to pH 8-9 withconcentrated NH₄OH while cooling in an ice bath. The resulting brownprecipitate is filtered off, and the filtrate is extracted with ether(4×60 mL). The combined ether extracts are washed with brine (4×60 mL),dried (Na₂SO₄), filtered, and evaporated to give a yellow/brown solid.This solid is combined with that from the above filtration and the wholeis triturated with cold ether to give the title compound as a lightbrown solid. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (1H, m), 7.21 (1H, m), 7.03(1H, m), 5.74 (2H, bs), 4.44 (2H, q, J 7.2, 6.9), 1.45 (3H, t, J 6.9).

F.3-(4-Fluorophenyl)-2-thioxo-2,3-dihydropyrido[3,2-d]pyrimidin-4(1H)-one

A mixture of ethyl 3-aminopyridine-2-carboxylate (2.0 g, 12.0 mmol) and4-fluorophenylisothiocyanate (1.84 g, 12.0 mmol) in 7 mL of anhydrouspyridine is stirred at 45° C. for 21 h. After cooling, the pyridine isevaporated in vacuo, and ice water is added to the residue. Theresulting mixture is slurried in EtOAc and filtered to give the titlecompound as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (1H, m),7.79 (2H, m), 7.33 (4H, m).

G. 2-Chloro-3-(4-fluorophenyl)pyrido[3,2-d]pyrimidin-4(3H)-one

A mixture of3-(4-fluorophenyl)-2-thioxo-2,3-dihydropyrido[3,2-d]pyrimidin-4(1H)-one(2.6 g, 9.5 mmol) in 30 mL of POCl₃ is heated to 135° C. and stirred for2 days. After cooling to RT, the excess POCl₃ is removed in vacuo, andthe residue is azeotroped twice with toluene. The resulting sticky brownoil/solid mix is dissolved in CH₂Cl₂ and neutralized to pH 7-8 withsaturated NaHCO₃. The layers are separated, and the CH₂Cl₂ layer isdried (Na₂SO₄), filtered, and evaporated to give a brown sticky solid.Purification by column chromatography (gradient from CH₂Cl₂ to 20%EtOAc/CH₂Cl₂) affords the title compound as an off-white solid. ¹H NMR(400 MHz, CDCl₃) δ 8.91 (1H, m), 8.05 (1H, m), 7.74 (1H, m), 7.28 (4H,m).

H. Acetic Formic Anhydride

Acetic anhydride (35.94 g, 352 mmol) and formic acid (16.20 g, 352 mmol)are added to a round bottomed flask, and heated at 55° C. for 3 h. Thereaction mixture is used in Example 1I without further purification.

I. Ethyl N-formyl-3-nitriloalaninate

Ethyl 3-nitriloalaninate (26.9 g, 210 mmol) is dissolved in anhydrousether (200 mL), and cooled in an ice/water bath. Acetic formic anhydride(prepared as a mixture as described above) is added dropwise. When theaddition is finished, the reaction mixture is allowed to warm to RT andstirred at RT overnight. Most volatiles are removed in vacuo, and theresidue solvents are removed by co-evaporation with toluene (100 mL×4).The red oil obtained precipitates upon scratching in ether, and theresulting solids are recrystallized in ether to give the title compoundas a white solid. ¹H NMR (400 MHz, CDCl₃) 8.32 (1H, s), 7.26 (1H, s),6.46 (1H, bs,), 5.56 (1H, d, J 7.8), 4.39 (2H, q), 1.37 (3H, t).

J. Ethyl 5-amino-1,3-thiazole-4-carboxylate

Ethyl N-formyl-3-nitriloalaninate (11.22 g, 71.86 mmol) is dissolved inanhydrous benzene (220 mL). Following the addition of Lawesson's reagent(14.53 g, 35.93 mmol), the suspension is refluxed for 24 h. Most of thesolvent is removed in vacuo, and the viscous red residue is absorbed onsilica gel and loaded to a silica gel column (elution solvent:EtOAc:hexanes=50:50). The title compound is obtained as yellow solids.¹H NMR (400 MHz, CDCl₃) 7.87 (1H, s), 7.26 (1 H, s), 6.01 (2H, broad s),4.38 (2H, q), 1.41 (3H, t).

K. 6-(4-Fluorophenyl)-5-mercapto[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-onehydrochloride

Ethyl 5-amino-1,3-thiazole-4-carboxylate (1.05 g, 6.10 mmol) and4-fluorophenyl isothiocyanate (0.93 g, 6.10 mmol) are added to pyridine(3.5 mL) and heated at 45° C. for 15 h. Most of the solvent is removedunder vacuum, and the resulting yellow solids are dissolved in CH₂Cl₂(150 mL) and washed with H₂O (20 mL×2) and brine (20 mL×2). The CH₂Cl₂phase is dried over MgSO₄, and the solvent is removed under reducedpressure. The resulting residue is treated with 1% NaOH solution (37 mL)and heated at 90° C. for 15 h. The reaction mixture is filtered and thefiltrate adjusted to pH 3 by the addition of concentrated HCl. Most ofthe water is removed under vacuum and the yellow solid which separatesis filtered and dried to give the title compound as a yellow solid. ¹HNMR (400 MHz, DMSO-d₆) 8.90 (1H, s), 7.31 (4H, m).

L. 5-Chloro-6-(4-fluorophenyl)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one

6-(4-Fluorophenyl)-5-mercapto[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-onehydrochloride (0.2 g, 0.633 mmol) is added to POCl₃ (10 mL), and theresulting solution is refluxed at 135° C. for 41 h. Most of thevolatiles are removed under reduced pressure and the residual solvent isco-evaporated with toluene (50 mL×3). The dark solid obtained isdissolved in CH₂Cl₂ (200 mL) and washed with saturated NaHCO₃ solution(100 mL×5), brine (50 mL×2), and dried over MgSO₄. After removingsolvent, silica gel column chromatography (EtOAc:hexanes=50:50) yieldsthe title compound as a yellow solid. ¹H NMR (400 MHz, CDCl₃) 8.88 (1H,s), 7.26 (4H, m).

M. 1-(4-Bromophenyl)-2-mercapto-9-methyl-1,9-dihydro-6H-purin-6-onehydrochloride

Ethyl 5-amino-1-methyl-1H-imidazole-4-carboxylate (3.50 g, 20.69 mmol)and 4-bromophenyl isothiocyanate (4.43 g, 20.69 mmol) are added topyridine (12 mL). The solution is heated at 45° C. for 3 h. Most of thesolvent is removed under vacuum and the resulting solid is dissolved inCH₂Cl₂ (300 mL). The CH₂Cl₂ solution is washed with water (30 mL×2),brine (30 mL×2), dried over MgSO₄, and concentrated in vacuo. Theresulting yellow solid is treated with 1% NaOH aqueous solution (125 mL)and heated at 90° C. for 18 h. The reaction mixture is filtered and thefiltrate adjusted pH to 3 by the addition of concentrated HCl. Most ofthe water is removed under reduced pressure. The resulting solid iscollected by filtration, the solid is azeotroped with toluene (30 mL×3)to give the title compound as a yellow solid. ¹H NMR (400 MHz, CD₃OD)8.10 (1H, s), 7.65 (2H, d), 7.14 (2H, d), 3.85 (3H, s).

N. 1-(4-Bromophenyl)-2-chloro-9-methyl-1,9-dihydro-6H-purin-6-one

1-(4-Bromophenyl)-2-mercapto-9-methyl-1,9-dihydro-6H-purin-6-onehydrochloride (2.05 g, 5.49 mmol) is added to POCl₃ (87 mL) and theresulting solution is refluxed at 135° C. for 38 h. Most volatiles areremoved under vacuum and the residual solvents are co-evaporated withtoluene (50 mL×3). The resulting dark solid is dissolved in CH₂Cl₂ (300mL), washed with saturated NaHCO₃ solution (100 mL×5), brine (50 mL×2),and dried over MgSO₄. After removing solvent in vacuo, silica gel columnchromatography (EtOAc:hexanes=50:50) gives the title compound as ayellow solid. ¹H NMR (400 MHz, CDCl₃) 7.51 (1H, s), 7.67 (2H, d), 7.14(2H, d), 3.83 (3H, s); m/z (ES⁺) 340.90 (M⁺).

Example 2 Synthesis of Representative 2-Phenoxy Pyrimidinone DerivativesA.1-(4-Bromophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one(Compound 1)

1-(4-Bromophenyl)-2-chloro-9-methyl-1,9-dihydro-6H-purin-6-one (150 mg,0.4417 mmol) and 2,3,4-trifluorophenol (130.8 mg, 0.8834 mmol) are addedto a vial, and the sealed mixture is heated with stirring at 140° C. for23 h. Silica gel column chromatography (MeOH:CH₂Cl₂=0.5:99.5) gives thetitle compound as a white solid. ¹H NMR (400 MHz, CDCl₃) 7.66 (3H, m),7.24 (2H, m), 7.00 (2H, m), 3.58 (3H, s). MS (M+1): 451.08; R_(T)=1.31min. The IC₅₀ determined as described in Example 6 is 100 nanomolar orless.

B.1-(4-Fluorophenyl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one(Compound 2)

A mixture of2-chloro-1-(4-fluorophenyl)-9-ethyl-1,9-dihydro-6H-purin-6-one (55.6 mg,0.20 mmol) and 3,4,5-trifluorophenol (0.40 mmol) is heated to 140° C.for 36 h. After cooling to RT, the crude mixture is purified bypreparative HPLC to give the title compound as a white solid. ¹H NMR(400 MHz, DMSO-d₆) 7.98 (1H, s), 7.55 (2H, m), 7.45 (2H, m), 7.34 (2H,m), 3.53 (3H, S). m/z=391.05 (M+1); R_(T)=0.78 min.

C.3-(4-Fluorophenyl)-2-[2-fluoro-3-(trifluoromethyl)phenoxy]pyrido[3,2-d]pyrimidin-4(3H)-one(Compound 3)

A mixture of 2-chloro-3-(4-fluorophenyl)pyrido[3,2-d]pyrimidin-4(3H)-one(55 mg, 0.20 mmol) and 2-fluoro-3-trifluoromethylphenol (50 μl, 0.40mmol) is heated to 140° C. for 18 hours. After cooling to RT, the crudemixture is purified by column chromatography (gradient from CH₂Cl₂ to20% EtOAc/CH₂Cl₂) to give the title compound as a white solid. ¹H NMR(400 MHz, DMSO-d₆) 8.71 (1H, m), 7.83 (2H, m), 7.70 (4H, m), 7.45 (3H,m). MS (M+1): 420.07; R_(T)=1.13 min.

D.5-(2,4-Difluorophenoxy)-6-(4-fluorophenyl)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one(Compound 4)

5-Chloro-6-(4-fluorophenyl)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one (0.2mmol) and 2.4-difluorophenol (0.4 mmol) are added to a vial, and thesealed mixture is heated with stirring at 140° C. for 48 h. Silica gelcolumn chromatography (MeOH:CH₂Cl₂=0.5:99.5) gives the title compound asa slightly yellow solid. ¹H NMR (400 MHz, CDCl₃) 8.70 (1H, s), 7.36 (2H,m), 7.26 (2H, m), 7.14 (1H, m), 6.94 (2H, m). MS (M+1): 376.03;R_(T)=1.33 min.

E.4-[9-Methyl-6-oxo-2-(2,3,4-trifluorophenoxy)-6,9-dihydro-1H-purin-1-yl]benzonitrile(Compound 5)

1-(4-Bromophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one(68 mg, 0.1507 mmol), Zn(CN)₂ (10.6 mg, 0.0904 mmol), Pd₂(dba)₃ (4.1 mg,0.0045 mmol) and 1,1′-bis(diphenylphosphino)ferrocene (5.0 mg, 0.0090mmol) are added to DMF (1.2 mL) and H₂O (0.012 mL). The mixture ispurged with N₂ for 3 min and then heated at 120° C. for 18 h. Themixture is passed through celite. Water (30 mL) is added and theresulting mixture is extracted with CH₂Cl₂ (50 mL×4). After drying theCH₂Cl₂ over MgSO₄, the solvent is removed in vacuo. The crude product ispurified by column chromatography (MeOH:CH₂Cl₂=2:98) to afford the titlecompound as a brown solid. ¹H NMR (400 MHz, CDCl₃) 7.86 (2H, d, J 8),7.65 (1H, s), 7.53 (2h, d, J 8), 6.95 (2H, m), 3.59 (3H, s). MS (M+1):398.03; R_(T)=1.22 min.

F.1-(6-Chloropyridin-3-yl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1H-purin-6(9H)-one(Compound 6) Step 1.2-Hydroxy-1-(6-chloropyridin-3-yl)-1,9-dihydro-6H-purin-6-one

Methyl 5-amino-1-methyl-1H-imidazole-4-carboxylate (1.0 g, 0.006 moles),2-chloro-5-isocyantopyridine (1.0 g, 0.006 moles) and DMAP (0.4 g, 0.003mole) are suspended in EtOAc (150 mL) and the resulting reaction mixtureis refluxed overnight. The reaction mixture is filtered, washed withEtOAc, and concentrated under vacuum. The white residue is suspended in1% aqueous NaOH (53 mL) and stirred at 90° C. for 20 h. The reactionmixture is neutralized with 6N HCl and extracted with DCM to afford thetitle compound.

Step 2. 2-Chloro-1-(6-chloropyridin-3-yl)-1,9-dihydro-6H-purin-6-one

2-Hydroxy-1-(6-chloropyridin-3-yl)-1,9-dihydro-6H-purin-6-one from theabove reaction is suspended in a large excess of phosphorous oxychloride(150 mL) and heated to 135° C. for 24 h. The reaction mixture is cooled,evaporated in vacuo, and additional toluene is added and evaporated (2×)under reduced pressure. The resulting sticky brown oil is dissolved inDCM (200 mL), and then neutralized with saturated NaHCO₃ (aqueous). Theaqueous layer is extracted with DCM (2×200 mL) and dried (MgSO₄). Thedried extract is filtered and concentrated under vacuum to afford crudeproduct as a light brown solid. The crude product is purified by flashcolumn chromatography using 1-2.5% MeOH/CH₂Cl₂ to afford the titlecompound as white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.33 (s, 1H), 7.78(s, 1H), 7.58 (d, J=6 Hz, 1H), 7.53 (d, J=6 Hz, 1H).

Step 3.1-(6-chloropyridin-3-yl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1H-purin-6(9H)-one(Compound 6)

1.0 M KO^(t)Bu in isopropanol (2 mL) is added to the solution of3,4,5-trifluorophenol (0.29 g, 1.86 mmol) and stirred for 20 minutes atRT. To the resulting phenoxide solution is added a solution of2-chloro-1-(6-chloropyridin-3-yl)-1,9-dihydro-6H-purin-6-one (0.5 g,1.69 mmol) in dimethylacetamide (3 mL) and the resulting mixture isstirred overnight at 50° C. The resulting reaction mixture is cooled,quenched with saturated NH₄Cl solution, extracted with DCM (2×50 mL) anddried (Na₂SO₄). The organic layer is filtered and concentrated undervacuum to afford the crude product as a light brown solid. The crudeproduct is further purified by flash column chromatography using 5%MeOH/CH₂Cl₂ to afford the title compound as white solid. ¹H NMR (300MHz, CDCl₃) δ 8.41 (s, 1H), 7.55-7.81 (m, 2H), 7.4 (brs, 1H), 6.71-7.02(m, 2H), 3.85 (s, 3H). MS (M+1): 408.04; R_(T)=1.48 min. The IC₅₀determined as described in Example 6 is 100 nanomolar or less.

G.5-(9-Methyl-6-oxo-2-(3,4,5-trifluorophenoxy)-6H-purin-1(9H)-yl)picolinonitrile(Compound 7)

1-(6-chloropyridin-3-yl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one(0.3 g, 0.73 mmol), Zn(CN)₂ (0.43 mg, 3.65 mmol), Pd₂(dba)₃ (0.07 g,0.073 mmol) and DPPF (0.04 g, 0.073 mmol) are added to DMF (3 mL). Themixture is purged with N₂ for 3 min and then heated at 120° C. for 18 h.Water (20 mL) is added and the resulting mixture is extracted with DCM(2×20 mL). The organic layer is passed through celite and Na₂SO₄ and thesolvent is removed in vacuo. The crude product is purified by columnchromatography on silica gel (MeOH:CH₂Cl₂=2:98) to afford the titlecompound as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.71 (s, 1H), 7.90(s, 2H), 7.68 (s, 1H), 7.25 (s, 2H), 6.85-6.89 (m, 2H), 3.65 (s, 3H, s).MS (M+1): 399.07; R_(T)=1.41 min. The IC₅₀ determined as described inExample 6 is 100 nanomolar or less.

Example 3 Additional Representative 2-Phenoxy Pyrimidinone Derivatives

Using routine modifications, the starting materials may be varied andadditional steps employed to produce other compounds provided herein.Compounds listed in Table I are prepared using such methods. In thecolumn labeled “IC₅₀” a * indicates that the IC₅₀ determined asdescribed in Example 6 is 100 nanomolar or less (i.e., the concentrationof such compounds that is required to provide a 50% decrease in thefluorescence response of cells exposed to one IC₅₀ of capsaicin is 100nanomolar or less). Mass spectroscopy data obtained as described aboveis presented as M+1 in the column headed “MS”, and retention times areprovided in the column headed “R_(T),” in minutes.

TABLE I Representative 2-Phenoxy Pyrimidinone Derivatives Compound NameMS R_(T) IC₅₀  8

1-(4-chlorophenyl)-9-methyl-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 406.99 1.27 *  9

9-ethyl-1-(4-fluorophenyl)-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 405.03 1.25 * 10

1-(4-chlorophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 407.00 1.28 * 11

9-ethyl-1-(4-fluorophenyl)-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 405.04 1.26 * 12

1-(4-fluorophenyl)-9-methyl-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 391.03 1.23 * 13

1-(4-fluorophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 391.04 1.24 * 14

1-(4-chlorophenyl)-2-(2,4- difluorophenoxy)-9-methyl-1,9-dihydro-6H-purin-6-one 389.02 1.25 * 15

2-(2,4-difluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one 387.06 1.24 * 16

2-(2,4-difluorophenoxy)-1-(4- fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one 373.03 1.22 * 17

2-(2,3-difluorophenoxy)-1-(4- fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one 373.05 1.22 * 18

2-(2,3-difluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one 387.05 1.24 * 19

2-(4-chloro-2-fluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one 403.00 1.28 * 13

2-(4-chloro-2-fluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H- purin-6-one 389.00 1.26 * 14

2-(4-chloro-2-fluorophenoxy)-1-(4-chlorophenyl)-9-methyl-1,9-dihydro-6H- purin-6-one 404.96 1.3  * 15

1-(4-fluorophenyl)-9-methyl-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 391.07 1.18 * 16

9-ethyl-1-(4-fluorophenyl)-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 405.09 1.11 * 17

1-(4-chlorophenyl)-9-methyl-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 407.04 1.3  * 18

2-(2,3-difluoro-4-methoxyphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H- purin-6-one 403.09 0.89 * 19

2-(2,3-difluoro-4-methoxyphenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H- purin-6-one 417.11 0.56 * 20

1-(4-fluorophenyl)-9-methyl-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 391.07 1.21 * 21

1-(4-chlorophenyl)-2-(2,3- difluorophenoxy)-9-methyl-1,9-dihydro-6H-purin-6-one 389.05 1.26 * 22

2-(2-chloro-4-fluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H- purin-6-one 389.05 1.22 * 23

2-(2-chloro-4-fluorophenoxy)-1-(4-chlorophenyl)-9-methyl-1,9-dihydro-6H- purin-6-one 405.02 0.6  * 24

1-(4-fluorophenyl)-9-methyl-2-(2,3,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 391.07 1.18 * 25

1-(4-chlorophenyl)-9-methyl-2-(2,3,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 407.04 0.62 * 26

9-ethyl-1-(4-fluorophenyl)-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 405.09 0.72 * 27

1-(4-chlorophenyl)-9-methyl-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 407.04 0.69 * 28

4-{[1-(4-chlorophenyl)-9-methyl-6-oxo-6,9-dihydro-1H-purin-2-yl]oxy}-2,3- difluorobenzonitrile 414.06 1.25 *29

2,3-difluoro-4-{[1-(4-fluorophenyl)-9-methyl-6-oxo-6,9-dihydro-1H-purin-2- yl]oxy}benzonitrile 398.07 1.22 *30

1-(4-chlorophenyl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6- one 407.04 1.39 * 31

2-(2,4-difluorophenoxy)-3-(4- fluorophenyl)pyrido[3,2-d]pyrimidin-4(3H)-one 370.11 1.26 * 32

3-(4-fluorophenyl)-2-(2,3,4- trifluorophenoxy)pyrido[3,2-d]pyrimidin-4(3H)-one 388.09 1.3  * 33

6-(4-fluorophenyl)-5-(2,3,4- trifluorophenoxy)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one 394.02 1.39 * 34

5-(2,3-difluorophenoxy)-6-(4-fluorophenyl)[1,3]thiazolo[5,4-d]pyrimidin- 7(6H)-one 376.02 1.27 * 35

6-(4-fluorophenyl)-5-(2,4,5- trifluorophenoxy)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one 394.02 0.66 * 36

6-(4-fluorophenyl)-5-(2,3,5- trifluorophenoxy)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one 394.01 1.29 * 37

6-(4-fluorophenyl)-5-(3,4,5- trifluorophenoxy)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one 394.02 1.41 * 38

5-(4-chloro-2-fluorophenoxy)-6-(4-fluorophenyl)[1,3]thiazolo[5,4-d]pyrimidin- 7(6H)-one 392.00 1.09 * 39

3-(4-fluorophenyl)-2-[3- (trifluoromethyl)phenoxy]pyrido[3,2-d]pyrimidin-4(3H)-one 402.08 1.44 40

2-[2-chloro-3-(trifluoromethyl)phenoxy]-3-(4-fluorophenyl)pyrido[3,2-d]pyrimidin- 4(3H)-one 436.04 0.51 41

6-(4-fluorophenyl)-5-[2-fluoro-3-(trifluoromethyl)phenoxy][1,3]thiazolo[5,4- d]pyrimidin-7(6H)-one 426.030.63 42

1-(4-chlorophenyl)-2-[2-fluoro-3-(trifluoromethyl)phenoxy]-9-methyl-1,9- dihydro-6H-purin-6-one 439.050.63 * 43

1-(4-fluorophenyl)-2-[2-fluoro-3-(trifluoromethyl)phenoxy]-9-methyl-1,9- dihydro-6H-purin-6-one 423.081.39 * 44

6-(4-fluorophenyl)-5-[3-fluoro-5-(trifluoromethyl)phenoxy][1,3]thiazolo[5,4- d]pyrimidin-7(6H)-one 426.030.51 * 45

5-[2-chloro-3-(trifluoromethyl)phenoxy]-6-(4-fluorophenyl)[1,3]thiazolo[5,4- d]pyrimidin-7(6H)-one 442.00 0.51 46

6-(4-fluorophenyl)-5-(3- (trifluoromethyl)phenoxy)thiazolo[5,4-d]pyrimidin-7(6H)-one 408.04 1.48 * 47

3-(4-fluorophenyl)-2-[3-fluoro-5- (trifluoromethyl)phenoxy]pyrido[3,2-d]pyrimidin-4(3H)-one 420.07 0.52 48

1-(4-fluorophenyl)-9-methyl-2-[3-(trifluoromethyl)phenoxy]-1,9-dihydro-6H- purin-6-one 405.09 0.54 * 49

1-(4-fluorophenyl)-2-[3-fluoro-5-(trifluoromethyl)phenoxy]-9-methyl-1,9- dihydro-6H-purin-6-one 423.081.36 * 50

3-(4-chlorophenyl)-7-methyl-2-(2,3,4-trifluorophenoxy)thieno[3,2-d]pyrimidin- 4(3H)-one 423.03 1.4  * 51

2-(4-chloro-2-fluorophenoxy)-3-(4- chlorophenyl)-7-methylthieno[3,2-d]pyrimidin-4(3H)-one 421.00 1.41 * 52

3-(4-chlorophenyl)-2-(2,4- difluorophenoxy)-7-methylthieno[3,2-d]pyrimidin-4(3H)-one 405.05 1.37 * 53

3-(4-fluorophenyl)-7-methyl-2-(2,3,4-trifluorophenoxy)thieno[3,2-d]pyrimidin- 4(3H)-one 405.07 1.4  * 54

2-(4-chloro-2-fluorophenoxy)-3-(4- fluorophenyl)-7-methylthieno[3,2-d]pyrimidin-4(3H)-one 405.02 0.65 * 55

3-(4-fluorophenyl)-7-methyl-2-(2,3,6-trifluorophenoxy)thieno[3,2-d]pyrimidin- 4(3H)-one 407.07 1.36 * 56

3-(4-fluorophenyl)-4-oxo-2-(2,3,4-trifluorophenoxy)-3,4-dihydrothieno[3,2- d]pyrimidine-7-carbonitrile417.99 1.31 57

3-(4-fluorophenyl)-2-(2-methoxyphenoxy)-7-methylthieno[3,2-d]pyrimidin-4(3H)-one 383.09 1.31 58

3-(4-fluorophenyl)-7-methyl-2-(2- methylphenoxy)thieno[3,2-d]pyrimidin-4(3H)-one 367.09 1.36 59

2-(2-ethylphenoxy)-3-(4-fluorophenyl)-7-methylthieno[3,2-d]pyrimidin-4(3H)-one 381.11 1.38 60

3-(4-fluorophenyl)-2-(2-isopropylphenoxy)-7-methylthieno[3,2-d]pyrimidin-4(3H)-one 395.12 1.39 61

2-(2,3-dimethylphenoxy)-3-(4- fluorophenyl)-7-methylthieno[3,2-d]pyrimidin-4(3H)-one 413.09 1.3  62

2-(2,6-dimethylphenoxy)-1-(4- fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one 365.14 1.28 63

2-(2,3-dimethylphenoxy)-1-(4- fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one 365.13 1.28 *

Example 4 VR1-Transfected Cells and Membrane Preparations

This Example illustrates the preparation of VR1-transfected cells andVR1-containing membrane preparations for use in capsaicin binding assays(Example 5).

A cDNA encoding full length human capsaicin receptor (SEQ ID NO:1, 2 or3 of U.S. Pat. No. 6,482,611) is subcloned in the plasmid pBK-CMV(Stratagene, La Jolla, Calif.) for recombinant expression in mammaliancells.

Human embryonic kidney (HEK293) cells are transfected with the pBK-CMVexpression construct encoding the full length human capsaicin receptorusing standard methods. The transfected cells are selected for two weeksin media containing G418 (400 μg/ml) to obtain a pool of stablytransfected cells. Independent clones are isolated from this pool bylimiting dilution to obtain clonal stable cell lines for use insubsequent experiments.

For radioligand binding experiments, cells are seeded in T175 cellculture flasks in media without antibiotics and grown to approximately90% confluency. The flasks are then washed with PBS and harvested in PBScontaining 5 mM EDTA. The cells are pelleted by gentle centrifugationand stored at −80° C. until assayed.

Previously frozen cells are disrupted with the aid of a tissuehomogenizer in ice-cold HEPES homogenization buffer (5 mM KCl 5, 5.8 mMNaCl, 0.75 mM CaCl₂, 2 mM MgCl₂, 320 mM sucrose, and 10 mM HEPES pH7.4). Tissue homogenates are first centrifuged for 10 minutes at 1000×g(4° C.) to remove the nuclear fraction and debris, and then thesupernatant from the first centrifugation is further centrifuged for 30minutes at 35,000×g (4° C.) to obtain a partially purified membranefraction. Membranes are resuspended in the HEPES homogenization bufferprior to the assay. An aliquot of this membrane homogenate is used todetermine protein concentration via the Bradford method (BIO-RAD ProteinAssay Kit, #500-0001, BIO-RAD, Hercules, Calif.).

Example 5 Capsaicin Receptor Binding Assay

This Example illustrates a representative assay of capsaicin receptorbinding that may be used to determine the binding affinity of compoundsfor the capsaicin (VR1) receptor.

Binding studies with [³H] Resiniferatoxin (RTX) are carried outessentially as described by Szallasi and Blumberg (1992) J. Pharmacol.Exp. Ter. 262:883-888. In this protocol, non-specific RTX binding isreduced by adding bovine alpha₁ acid glycoprotein (100 μg per tube)after the binding reaction has been terminated.

[³H] RTX (37 Ci/mmol) is synthesized by and obtained from the ChemicalSynthesis and Analysis Laboratory, National Cancer Institute-FrederickCancer Research and Development Center, Frederick, Md. [³H] RTX may alsobe obtained from commercial vendors (e.g., Amersham Pharmacia Biotech,Inc.; Piscataway, N.J.).

The membrane homogenate of Example 4 is centrifuged as before andresuspended to a protein concentration of 333 μg/ml in homogenizationbuffer. Binding assay mixtures are set up on ice and contain [³H]RTX(specific activity 2200 mCi/ml), 2 μl non-radioactive test compound,0.25 mg/ml bovine serum albumin (Cohn fraction V), and 5×10⁴-1×10⁵VR1-transfected cells. The final volume is adjusted to 500 μl (forcompetition binding assays) or 1,000 μl (for saturation binding assays)with the ice-cold HEPES homogenization buffer solution (pH 7.4)described above. Non-specific binding is defined as that occurring inthe presence of 1 μM non-radioactive RTX (Alexis Corp.; San Diego,Calif.). For saturation binding, [³H]RTX is added in the concentrationrange of 7-1,000 pM, using 1 to 2 dilutions. Typically 11 concentrationpoints are collected per saturation binding curve.

Competition binding assays are performed in the presence of 60 pM[³H]RTX and various concentrations of test compound. The bindingreactions are initiated by transferring the assay mixtures into a 37° C.water bath and are terminated following a 60 minute incubation period bycooling the tubes on ice. Membrane-bound RTX is separated from free, aswell as any alpha₁-acid glycoprotein-bound RTX, by filtration ontoWALLAC glass fiber filters (PERKIN-ELMER, Gaithersburg, Md.) which arepre-soaked with 1.0% PEI (polyethyleneimine) for 2 hours prior to use.Filters are allowed to dry overnight then counted in a WALLAC 1205 BETAPLATE counter after addition of WALLAC BETA SCINT scintillation fluid.

Equilibrium binding parameters are determined by fitting the allostericHill equation to the measured values with the aid of the computerprogram FIT P (Biosoft, Ferguson, Mo.) as described by Szallasi, et al.(1993) J. Pharmacol. Exp. Ther. 266:678-683. Compounds provided hereingenerally exhibit K_(i) values for capsaicin receptor of less than 1 μM,100 nM, 50 nM, 25 nM, 10 nM, or 1 nM in this assay.

Example 6 Calcium Mobilization Assay

This Example illustrates representative calcium mobilization assays foruse in evaluating test compounds for agonist and antagonist activity.

Cells transfected with expression plasmids (as described in Example 4)and thereby expressing human capsaicin receptor are seeded and grown to70-90% confluency in FALCON black-walled, clear-bottomed 96-well plates(#3904, BECTON-DICKINSON, Franklin Lakes, N.J.). The culture medium isemptied from the 96 well plates and FLUO-3 AM calcium sensitive dye(Molecular Probes, Eugene, Oreg.) is added to each well (dye solution: 1mg FLUO-3 AM, 440 μL DMSO and 440 μl 20% pluronic acid in DMSO, diluted1:250 in Krebs-Ringer HEPES (KRH) buffer (25 mM HEPES, 5 mM KCl, 0.96 mMNaH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂, 5 mM glucose, 1 mM probenecid, pH 7.4),50 μl diluted solution per well). Plates are covered with aluminum foiland incubated at 37° C. for 1-2 hours in an environment containing 5%CO₂. After the incubation, the dye is emptied from the plates, and thecells are washed once with KRH buffer, and resuspended in KRH buffer.

Determination of Capsaicin EC₅₀

To measure the ability of a test compound to agonize or antagonize acalcium mobilization response in cells expressing capsaicin receptors tocapsaicin or other vanilloid agonist, the EC₅₀ of the agonist capsaicinis first determined. An additional 20 μl of KRH buffer and 1 μl DMSO isadded to each well of cells, prepared as described above. 100 μlcapsaicin in KRH buffer is automatically transferred by the FLIPRinstrument to each well. Capsaicin-induced calcium mobilization ismonitored using either FLUOROSKAN ASCENT (Labsystems; Franklin, Mass.)or FLIPR (fluorometric imaging plate reader system; Molecular Devices,Sunnyvale, Calif.) instruments. Data obtained between 30 and 60 secondsafter agonist application are used to generate an 8-point concentrationresponse curve, with final capsaicin concentrations of 1 nM to 3 μM.KALEIDAGRAPH software (Synergy Software, Reading, Pa.) is used to fitthe data to the equation:y=a*(1/(1+(b/x)^(c)))to determine the 50% excitatory concentration (EC₅₀) for the response.In this equation, y is the maximum fluorescence signal, x is theconcentration of the agonist or antagonist (in this case, capsaicin), ais the E_(max), b corresponds to the EC₅₀ value and c is the Hillcoefficient.Determination of Agonist Activity

Test compounds are dissolved in DMSO, diluted in KRH buffer, andimmediately added to cells prepared as described above. 100 nM capsaicin(an approximate EC₉₀ concentration) is also added to cells in the same96-well plate as a positive control. The final concentration of testcompounds in the assay wells is between 0.1 nM and 5 μM.

The ability of a test compound to act as an agonist of the capsaicinreceptor is determined by measuring the fluorescence response of cellsexpressing capsaicin receptors elicited by the compound as function ofcompound concentration. This data is fit as described above to obtainthe EC₅₀, which is generally less than 1 micromolar, preferably lessthan 100 nM, and more preferably less than 10 nM. The extent of efficacyof each test compound is also determined by calculating the responseelicited by a concentration of test compound (typically 1 μM) relativeto the response elicited by 100 nM capsaicin. This value, called Percentof Signal (POS), is calculated by the following equation:POS=100*test compound response/100 nM capsaicin response

This analysis provides quantitative assessment of both the potency andefficacy of test compounds as human capsaicin receptor agonists.Agonists of the human capsaicin receptor generally elicit detectableresponses at concentrations less than 100 μM, or preferably atconcentrations less than 1 μM, or most preferably at concentrations lessthan 10 nM. Extent of efficacy at human capsaicin receptor is preferablygreater than 30 POS, more preferably greater than 80 POS at aconcentration of 1 μM. Certain agonists are essentially free ofantagonist activity as demonstrated by the absence of detectableantagonist activity in the assay described below at compoundconcentrations below 4 nM, more preferably at concentrations below 10 μMand most preferably at concentrations less than or equal to 100 μm.

Determination of Antagonist Activity

Test compounds are dissolved in DMSO, diluted in 20 μl KRH buffer sothat the final concentration of test compounds in the assay well isbetween 1 μM and 5 μM, and added to cells prepared as described above.The 96 well plates containing prepared cells and test compounds areincubated in the dark, at room temperature for 0.5 to 6 hours. It isimportant that the incubation not continue beyond 6 hours. Just prior todetermining the fluorescence response, 100 μl capsaicin in KRH buffer attwice the EC₅₀ concentration determined as described above isautomatically added by the FLIPR instrument to each well of the 96 wellplate for a final sample volume of 200 μl and a final capsaicinconcentration equal to the EC₅₀. The final concentration of testcompounds in the assay wells is between 1 μM and 5 μM. Antagonists ofthe capsaicin receptor decrease this response by at least about 20%,preferably by at least about 50%, and most preferably by at least 80%,as compared to matched control (i.e., cells treated with capsaicin attwice the EC₅₀ concentration in the absence of test compound), at aconcentration of 10 micromolar or less, preferably 1 micromolar or less.The concentration of antagonist required to provide a 50% decrease,relative to the response observed in the presence of capsaicin andwithout antagonist, is the IC₅₀ for the antagonist, and is preferablybelow 1 micromolar, 100 nanomolar, 10 nanomolar or 1 nanomolar.

The data is analyzed as follows. First, the average maximum relativefluorescent unit (RFU) response from the negative control wells (noagonist) is subtracted from the maximum response detected for each ofthe other experimental wells. Second, average maximum RFU response iscalculated for the positive control wells (agonist wells). Then, percentinhibition for each compound tested is calculated using the equation:Percent Inhibition=100−100×(Peak Signal in Test Cells/Peak Signal inControl Cells)The % inhibition data is plotted as a function of test compoundconcentration and test compound IC₅₀ is determined using, for example,KALEIDAGRAPH software (Synergy Software, Reading, Pa.) best fit of thedata to the equation:y=m ₁*(1/(1+(m ₂ /m ₀)^(m3)))where y is the percent inhibition, m₀ is the concentration of theagonist, m₁ is the maximum RFU, m₂ corresponds to the test compound IC₅₀(the concentration required to provide a 50% decrease, relative to theresponse observed in the presence of agonist and without antagonist) andm₃ is the Hill coefficient. Alternatively, test compound IC₅₀ isdetermined using a linear regression in which x is ln(concentration oftest compound) and y is ln(percent inhibition/(100−percent inhibition).Data with a percent inhibition that is greater than 90% or less than 15%are rejected and are not used in the regression. The IC₅₀ calculated inthis fashion is e^((−intercept/slope)).

Certain preferred VR1 modulators are antagonists that are essentiallyfree of agonist activity as demonstrated by the absence of detectableagonist activity in the assay described above at compound concentrationsbelow 4 nM, more preferably at concentrations below 10 μM and mostpreferably at concentrations less than or equal to 100 μM.

Example 7 Dorsal Root Ganglion Cell Assay

This Example illustrates a representative dorsal root ganglian cellassay for evaluating VR1 antagonist or agonist activity of a compound.

DRG are dissected from neonatal rats, dissociated and cultured usingstandard methods (Aguayo and White (1992) Brain Research 570:61-67).After 48 hour incubation, cells are washed once and incubated for 30-60minutes with the calcium sensitive dye Fluo 4 AM (2.5-10 ug/ml; TefLabs,Austin, Tex.). Cells are then washed once. Addition of capsaicin to thecells results in a VR1-dependent increase in intracellular calciumlevels which is monitored by a change in Fluo-4 fluorescence with afluorometer. Data are collected for 60-180 seconds to determine themaximum fluorescent signal.

For antagonist assays, various concentrations of compound are added tothe cells. Fluorescent signal is then plotted as a function of compoundconcentration to identify the concentration required to achieve a 50%inhibition of the capsaicin-activated response, or IC₅₀. Antagonists ofthe capsaicin receptor preferably have an IC₅₀ below 1 micromolar, 100nanomolar, 10 nanomolar or 1 nanomolar. For agonist assays, variousconcentrations of compound are added to the cells without the additionof capsaicin. Compounds that are capsaicin receptor agonists result in aVR1-dependent increase in intracellular calcium levels which ismonitored by a change in Fluo-4 fluorescence with a fluorometer. TheEC₅₀, or concentration required to achieve 50% of the maximum signal fora capsaicin-activated response, is preferably below 1 micromolar, below100 nanomolar or below 10 nanomolar.

Example 8 Animal Models for Determining Pain Relief

This Example illustrates representative methods for assessing the degreeof pain relief provided by a compound.

A. Pain Relief Testing

The following methods may be used to assess pain relief.

Mechanical Allodynia

Mechanical allodynia (an abnormal response to an innocuous stimulus) isassessed essentially as described by Chaplan et al. (1994) J. Neurosci.Methods 53:55-63 and Tal and Eliav (1998) Pain 64(3):511-518. A seriesof von Frey filaments of varying rigidity (typically 8-14 filaments in aseries) are applied to the plantar surface of the hind paw with justenough force to bend the filament. The filaments are held in thisposition for no more than three seconds or until a positive allodynicresponse is displayed by the rat. A positive allodynic response consistsof lifting the affected paw followed immediately by licking or shakingof the paw. The order and frequency with which the individual filamentsare applied are determined by using Dixon up-down method. Testing isinitiated with the middle hair of the series with subsequent filamentsbeing applied in consecutive fashion, ascending or descending, dependingon whether a negative or positive response, respectively, is obtainedwith the initial filament.

Compounds are effective in reversing or preventing mechanicalallodynia-like symptoms if rats treated with such compounds requirestimulation with a Von Frey filament of higher rigidity strength toprovoke a positive allodynic response as compared to control untreatedor vehicle treated rats. Alternatively, or in addition, testing of ananimal in chronic pain may be done before and after compoundadministration. In such an assay, an effective compound results in anincrease in the rigidity of the filament needed to induce a responseafter treatment, as compared to the filament that induces a responsebefore treatment or in an animal that is also in chronic pain but isleft untreated or is treated with vehicle. Test compounds areadministered before or after onset of pain. When a test compound isadministered after pain onset, testing is performed 10 minutes to threehours after administration.

Mechanical Hyperalgesia

Mechanical hyperalgesia (an exaggerated response to painful stimulus) istested essentially as described by Koch et al. (1996) Analgesia2(3):157-164. Rats are placed in individual compartments of a cage witha warmed, perforated metal floor. Hind paw withdrawal duration (i.e.,the amount of time for which the animal holds its paw up before placingit back on the floor) is measured after a mild pinprick to the plantarsurface of either hind paw.

Compounds produce a reduction in mechanical hyperalgesia if there is astatistically significant decrease in the duration of hindpawwithdrawal. Test compound may be administered before or after onset ofpain. For compounds administered after pain onset, testing is performed10 minutes to three hours after administration.

Thermal Hyperalgesia

Thermal hyperalgesia (an exaggerated response to noxious thermalstimulus) is measured essentially as described by Hargreaves et al.(1988) Pain. 32(1):77-88. Briefly, a constant radiant heat source isapplied the animals' plantar surface of either hind paw. The time towithdrawal (i.e., the amount of time that heat is applied before theanimal moves its paw), otherwise described as thermal threshold orlatency, determines the animal's hind paw sensitivity to heat.

Compounds produce a reduction in thermal hyperalgesia if there is astatistically significant increase in the time to hindpaw withdrawal(i.e., the thermal threshold to response or latency is increased). Testcompound may be administered before or after onset of pain. Forcompounds administered after pain onset, testing is performed 10 minutesto three hours after administration.

B. Pain Models

Pain may be induced using any of the following methods, to allow testingof analgesic efficacy of a compound. In general, compounds providedherein result in a statistically significant reduction in pain asdetermined by at least one of the previously described testing methods,using male SD rats and at least one of the following models.

Acute Inflammatory Pain Model

Acute inflammatory pain is induced using the carrageenan modelessentially as described by Field et al. (1997) Br. J. Pharmacol.121(8):1513-1522. 100-200 μl of 1-2% carrageenan solution is injectedinto the rats' hind paw. Three to four hours following injection, theanimals' sensitivity to thermal and mechanical stimuli is tested usingthe methods described above. A test compound (0.01 to 50 mg/kg) isadministered to the animal, prior to testing, or prior to injection ofcarrageenan. The compound can be administered orally or through anyparenteral route, or topically on the paw. Compounds that relieve painin this model result in a statistically significant reduction inmechanical allodynia and/or thermal hyperalgesia.

Chronic Inflammatory Pain Model

Chronic inflammatory pain is induced using one of the followingprotocols:

-   -   1. Essentially as described by Bertorelli et al. (1999) Br. J.        Pharmacol. 128(6):1252-1258, and Stein et al. (1998) Pharmacol.        Biochem. Behav. 31(2):455-51, 200 μl Complete Freund's Adjuvant        (0.1 mg heat killed and dried M. Tuberculosis) is injected to        the rats' hind paw: 100 μl into the dorsal surface and 100 μl        into the plantar surface.    -   2. Essentially as described by Abbadie et al. (1994) J Neurosci.        14(10):5865-5871 rats are injected with 150 μl of CFA (1.5 mg)        in the tibio-tarsal joint.

Prior to injection with CFA in either protocol, an individual baselinesensitivity to mechanical and thermal stimulation of the animals' hindpaws is obtained for each experimental animal.

Following injection of CFA, rats are tested for thermal hyperalgesia,mechanical allodynia and mechanical hyperalgesia as described above. Toverify the development of symptoms, rats are tested on days 5, 6, and 7following CFA injection. On day 7, animals are treated with a testcompound, morphine or vehicle. An oral dose of morphine of 1-5 mg/kg issuitable as positive control. Typically, a dose of 0.01-50 mg/kg of testcompound is used. Compounds can be administered as a single bolus priorto testing or once or twice or three times daily, for several days priorto testing. Drugs are administered orally or through any parenteralroute, or applied topically to the animal.

Results are expressed as Percent Maximum Potential Efficacy (MPE). 0%MPE is defined as analgesic effect of vehicle, 100% MPE is defined as ananimal's return to pre-CFA baseline sensitivity. Compounds that relievepain in this model result in a MPE of at least 30%.

Chronic Neuropathic Pain Model

Chronic neuropathic pain is induced using the chronic constrictioninjury (CCI) to the rat's sciatic nerve essentially as described byBennett and Xie (1988) Pain 33:87-107. Rats are anesthetized (e.g. withan intraperitoneal dose of 50-65 mg/kg pentobarbital with additionaldoses administered as needed). The lateral aspect of each hind limb isshaved and disinfected. Using aseptic technique, an incision is made onthe lateral aspect of the hind limb at the mid thigh level. The bicepsfemoris is bluntly dissected and the sciatic nerve is exposed. On onehind limb of each animal, four loosely tied ligatures are made aroundthe sciatic nerve approximately 1-2 mm apart. On the other side thesciatic nerve is not ligated and is not manipulated. The muscle isclosed with continuous pattern and the skin is closed with wound clipsor sutures. Rats are assessed for mechanical allodynia, mechanicalhyperalgesia and thermal hyperalgesia as described above.

Compounds that relieve pain in this model result in a statisticallysignificant reduction in mechanical allodynia, mechanical hyperalgesiaand/or thermal hyperalgesia when administered (0.01-50 mg/kg, orally,parenterally or topically) immediately prior to testing as a singlebolus, or for several days: once or twice or three times daily prior totesting.

What is claimed is:
 1. A method for treating pain in a patient,comprising administering to a patient suffering from pain atherapeutically effective amount of at least one compound of theformula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

represents

wherein R₂ is hydrogen, C₁-C₄alkyl, C₁-C₄haloalkyl or C₃-C₅cycloalkyl;Ar is phenyl or a 5- or 6-membered heteroaryl, each of which issubstituted with from 0 to 4 substituents that are independently chosenfrom halogen, cyano, amino, nitro, C₁-C₆alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆alkoxy,C₁-C₆haloalkoxy, (C₃-C₇cycloalkyl)C₀-C₄alkyl, and mono- ordi-(C₁-C₆alkyl)amino; and R₃ represents from 0 to 4 substituents thatare independently chosen from halogen, hydroxy, cyano, amino, nitro,C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆haloalkyl,C₁-C₆hydroxyalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,(C₃-C₇cycloalkyl)C₀-C₄alkyl, mono- or di-(C₁-C₆alkyl)amino, and mono- ordi-(C₁-C₆alkyl)aminosulfonyl, and thereby alleviating pain in thepatient.
 2. A method for treating itch in a patient, comprisingadministering to a patient a therapeutically effective amount of acompound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

represents

wherein R₂ is hydrogen, C₁-C₄alkyl, C₁-C₄haloalkyl or C₃-C₅cycloalkyl;Ar is phenyl or a 5- or 6-membered heteroaryl, each of which issubstituted with from 0 to 4 substituents that are independently chosenfrom halogen, cyano, amino, nitro, C₁-C₆alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆alkoxy,C₁-C₆haloalkoxy, (C₃-C₇cycloalkyl)C₀-C₄alkyl, and mono- ordi-(C₁-C₆alkyl)amino; and R₃ represents from 0 to 4 substituents thatare independently chosen from halogen, hydroxy, cyano, amino, nitro,C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆haloalkyl,C₁-C₆hydroxyalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,(C₃-C₇cycloalkyl)C₀-C₄alkyl, mono- or di-(C₁-C₆alkyl)amino, and mono- ordi-(C₁-C₆alkyl)aminosulfonyl, and thereby alleviating itch in thepatient.
 3. A method for treating cough or hiccup in a patient,comprising administering to a patient a therapeutically effective amountof a compound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

represents

wherein R₂ is hydrogen, C₁-C₄alkyl, C₁-C₄haloalkyl or C₃-C₅cycloalkyl;Ar is phenyl or a 5- or 6-membered heteroaryl, each of which issubstituted with from 0 to 4 substituents that are independently chosenfrom halogen, cyano, amino, nitro, C₁-C₆alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆alkoxy,C₁-C₆haloalkoxy, (C₃-C₇cycloalkyl)C₀-C₄alkyl, and mono- ordi-(C₁-C₆alkyl)amino; and R₃ represents from 0 to 4 substituents thatare independently chosen from halogen, hydroxy, cyano, amino, nitro,C₁-C₆alkyl, C₂-C₆alkenyl, C₂-₆alkynyl, C₁-C₆haloalkyl,C₁-C₆hydroxyalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,(C₃-C₇cycloalkyl)C₀-C₄alkyl, mono- or di-(C₁-C₆alkyl)amino, and mono- ordi-(C₁-C₆alkyl)aminosulfonyl, and thereby alleviating cough or hiccup inthe patient.
 4. A method for treating urinary incontinence or overactivebladder in a patient, comprising administering to a patient atherapeutically effective amount of a compound of the formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

represents

wherein R₂ is hydrogen, C₁-C₄haloalkyl or C₃-C₅cycloalkyl; Ar is phenylor a 5- or 6-membered heteroaryl, each of which is substituted with from0 to 4 substituents that are independently chosen from halogen, cyano,amino, nitro, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆haloalkyl,C₁-C₆hydroxyalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,(C₃-C₇cycloalkyl)C₀-C₄alkyl, and mono- or di-(C₁-C₆alkyl)amino; and R₃represents from 0 to 4 substituents that are independently chosen fromhalogen, hydroxy, cyano, amino, nitro, C₁-C₆alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆alkoxy,C₁-C₆haloalkoxy, (C₃-C₇cycloalkyl)C₀-C₄alkyl, mono- ordi-(C₁-C₆alkyl)amino, and mono- or di-(C₁-C₆alkyl)aminosulfonyl, andthereby alleviating urinary incontinence or overactive bladder in thepatient.
 5. A method for treating pain in a patient, comprisingadministering to a patient suffering from pain a therapeuticallyeffective amount of a combination of (i) at least one compound of theformula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

represents

wherein R₂ is hydrogen, C₁-C₄alkyl, C₁-C₄haloalkyl or C₃-C₅cycloalkyl;Ar is phenyl or a 5- or 6-membered heteroaryl, each of which issubstituted with from 0 to 4 substituents that are independently chosenfrom halogen, cyano, amino, nitro, C₁-C₆alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₁-C₆haloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆alkoxy,C₁-C₆haloalkoxy, (C₃-C₇cycloalkyl)C₀-C₄alkyl, and mono- ordi-(C₁-C₆alkyl)amino; and R₃ represents from 0 to 4 substituents thatare independently chosen from halogen, hydroxy, cyano, amino, nitro,C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆haloalkyl,C₁-C₆hydroxyalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,(C₃-C₇cycloalkyl)C₀-C₄alkyl, mono- or di-(C₁-C₆alkyl)amino, and mono- ordi-(C₁-C₆alkyl)aminosulfonyl, and (ii) ibuprofen, and therebyalleviating pain in the patient.
 6. The method of claim 1, wherein, inthe compound or salt or hydrate thereof, R₃ represents from 1to 3substituents independently chosen from halogen, cyano, C₁-C₆alkyl,C₁-C₆haloalkyl and C₁-C₆alkoxy.
 7. The method of claim 1, wherein thecompound or salt or hydrate thereof has the formula:

wherein: R₂ is hydrogen, C₁-C₄alkyl, C₁-C₄haloalkyl or C₃- C₅cycloalkyl;R₃ represents from 1to 3 substituents independently chosen from halogen,cyano, C₁-C₄alkyl, C₁-C₄haloalkyl and C₁-C₄alkoxy; and R₅ is halogen orCN.
 8. The method of claim 1, wherein the compound is:1-(4-bromophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-2-(2,3-difluorophenoxy)-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-2-(2,4-difluorophenoxy)-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-2-[2-fluoro-3-(trifluoromethyl)phenoxy]-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,3,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-2-[2-fluoro-3-(trifluoromethyl)phenoxy]-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-2-[3-fluoro-5-(trifluoromethyl)phenoxy]-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,3,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-[3-(trifluoromethyl)phenoxy]-1,9-dihydro-6H-purin-6-one;1-(6-chloropyridin-3-yl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1H-purin-6(9H)-one;2-(2,3-difluoro-4-methoxyphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,3-difluoro-4-methoxyphenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2-(2,3-difluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,3-difluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2-(2,3-dimethylphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,4-difluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,4-difluorophenoxy)-3-(4-fluorophenyl)pyrido[3,2-d]pyrimidin-4(3H)-one;2-(2,4-difluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2-(2,6-dimethylphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2-chloro-4-fluorophenoxy)-1-(4-chlorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2-chloro-4-fluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(4-chloro-2-fluorophenoxy)-1-(4-chlorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(4-chloro-2-fluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(4-chloro-2-fluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2,3-difluoro-4-{[1-(4-fluorophenyl)-9-methyl-6-oxo-6,9-dihydro-1H-purin-2-yl]oxy}benzonitrile;4-{([1-(4-chlorophenyl)-9-methyl-6-oxo-6,9-dihydro-1H-purin-2-yl]oxy}-2,3-difluorobenzonitrile;5-(9-Methyl-6-oxo-2-(3,4,5-trifluorophenoxy)-6H-purin-1(9H)-yl)picolinonitrile;5-[2-chloro-3-(trifluoromethyl)phenoxy]-6-(4-fluorophenyl)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one;9-ethyl-1-(4-fluorophenyl)-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;9-ethyl-1-(4-fluorophenyl)-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;9-ethyl-1-(4-fluorophenyl)-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;or9-ethyl-1-(4-fluorophenyl)-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one.9. The method of claim 2, wherein, in the compound or salt or hydratethereof, R₃ represents from 1to 3 substituents independently chosen fromhalogen, cyano, C₁-C₆alkyl, C₁-C₆haloalkyl and C₁-C₆alkoxy.
 10. Themethod of claim 2, wherein the compound or salt or hydrate thereof hasthe formula:

wherein: R₂ is hydrogen, C₁-C₄alkyl, C₁-C₄haloalkyl or C₃-C₅cycloalkyl;R₃ represents from 1to 3 substituents independently chosen from halogen,cyano, C₁-C₄alkyl, C₁-C₄haloalkyl and C₁-C₄alkoxy; and R₅ is halogen orCN.
 11. The method of claim 2, wherein the compound is:1-(4-bromophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-2-(2,3-difluorophenoxy)-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-2-(2,4-difluorophenoxy)-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-2-[2-fluoro-3-(trifluoromethyl)phenoxy]-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,3,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-2-[2-fluoro-3-(trifluoromethyl)phenoxy]-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-2-[3-fluoro-5-(trifluoromethyl)phenoxy]-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,3,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-[3-(trifluoromethyl)phenoxy]-1,9-dihydro-6H-purin-6-one;1-(6-chloropyridin-3-yl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1H-purin-6(9H)-one;2-(2,3-difluoro-4-methoxyphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,3-difluoro-4-methoxyphenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2-(2,3-difluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,3-difluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2-(2,3-dimethylphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,4-difluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,4-difluorophenoxy)-3-(4-fluorophenyl)pyrido[3,2-d]pyrimidin-4(3H)-one;2-(2,4-difluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2-(2,6-dimethylphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2-chloro-4-fluorophenoxy)-1-(4-chlorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2-chloro-4-fluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(4-chloro-2-fluorophenoxy)-1-(4-chlorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(4-chloro-2-fluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(4-chloro-2-fluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2,3-difluoro-4-{([1-(4-fluorophenyl)-9-methyl-6-oxo-6,9-dihydro-1H-purin-2-yl]oxy}benzonitrile;4-{([1-(4-chlorophenyl)-9-methyl-6-oxo-6,9-dihydro-1H-purin-2-yl]oxy}-2,3-difluorobenzonitrile;5-(9-Methyl-6-oxo-2-(3,4,5-trifluorophenoxy)-6H-purin-1(9H)-yl)picolinonitrile;5-[2-chloro-3-(trifluoromethyl)phenoxy]-6-(4-fluorophenyl)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one;9-ethyl-1-(4-fluorophenyl)-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;9-ethyl-1-(4-fluorophenyl)-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;9-ethyl-1-(4-fluorophenyl)-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;or9-ethyl-1-(4-fluorophenyl)-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one.12. The method of claim 4, wherein, in the compound or salt or hydratethereof, R₃ represents from 1to 3 substituents independently chosen fromhalogen, cyano, C₁-C₆alkyl, C₁-C₆haloalkyl and C₁-C₆alkoxy.
 13. Themethod of claim 4, wherein the compound or salt or hydrate thereof hasthe formula:

wherein: R₂ is hydrogen, C₁-C₄alkyl, C₁-C₄haloalkyl or C₃-C₅cycloalkyl;R₃ represents from 1to 3 substituents independently chosen from halogen,cyano, C₁-C₄alkyl, C₁-C₄haloalkyl and C₁-C₄alkoxy; and R₅ is halogen orCN.
 14. The method of claim 4, wherein the compound is:1-(4-bromophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-2-(2,3-difluorophenoxy)-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-2-(2,4-difluorophenoxy)-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-2-[2-fluoro-3-(trifluoromethyl)phenoxy]-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,3,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-chlorophenyl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-2-[2-fluoro-3-(trifluoromethyl)phenoxy]-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-2-[3-fluoro-5-(trifluoromethyl)phenoxy]-9-methyl-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,3,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;1-(4-fluorophenyl)-9-methyl-2-[3-(trifluoromethyl)phenoxy]-1,9-dihydro-6H-purin-6-one;1-(6-chloropyridin-3-yl)-9-methyl-2-(3,4,5-trifluorophenoxy)-1H-purin-6(9H)-one;2-(2,3-difluoro-4-methoxyphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,3-difluoro-4-methoxyphenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2-(2,3-difluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,3-difluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2-(2,3-dimethylphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,4-difluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2,4-difluorophenoxy)-3-(4-fluorophenyl)pyrido[3,2-d]pyrimidin-4(3H)-one;2-(2,4-difluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2-(2,6-dimethylphenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2-chloro-4-fluorophenoxy)-1-(4-chlorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(2-chloro-4-fluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(4-chloro-2-fluorophenoxy)-1-(4-chlorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(4-chloro-2-fluorophenoxy)-1-(4-fluorophenyl)-9-methyl-1,9-dihydro-6H-purin-6-one;2-(4-chloro-2-fluorophenoxy)-9-ethyl-1-(4-fluorophenyl)-1,9-dihydro-6H-purin-6-one;2,3-difluoro-4-{([1-(4-fluorophenyl)-9-methyl-6-oxo-6,9-dihydro-1H-purin-2-yl]oxy}benzonitrile;4-{([1-(4-chlorophenyl)-9-methyl-6-oxo-6,9-dihydro-1H-purin-2-yl]oxy}-2,3-difluorobenzonitrile;5-(9-Methyl-6-oxo-2-(3,4,5-trifluorophenoxy)-6H-purin-1(9H)-yl)picolinonitrile;5-[2-chloro-3-(trifluoromethyl)phenoxy]-6-(4-fluorophenyl)[1,3]thiazolo[5,4-d]pyrimidin-7(6H)-one;9-ethyl-1-(4-fluorophenyl)-2-(2,3,4-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;9-ethyl-1-(4-fluorophenyl)-2-(2,3,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;9-ethyl-1-(4-fluorophenyl)-2-(2,4,5-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one;or9-ethyl-1-(4-fluorophenyl)-2-(2,4,6-trifluorophenoxy)-1,9-dihydro-6H-purin-6-one.